Chemically inducible cucumber mosaic virus protein expression system

ABSTRACT

The invention relates to a novel chemically inducible plant viral amplicon (CMViva) expression system that permits controllable, high level expression of foreign genes in plant hosts. This system employs agro-infiltration of plants to provide a transient production of a protein of interest, such as a human blood protein. This system provides a major advantage over existing plant expression systems because it allows for consistent expression of foreign or heterologous proteins in plant hosts.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Phase patent application of PCT/US2007/020712,filed Sep. 24, 2007, which claims the benefit of U.S. ProvisionalApplication No. 60/846,704, filed on Sep. 22, 2006, all of which isincorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with Government support under Grant (orContract) No. BES-0214527 awarded by the National Science Foundation andNSF REU. The US Government has certain rights in the invention.

FIELD OF THE INVENTION

This invention is in the field of plant breeding and plant molecularbiology. In particular, this invention relates to a novel chemicallyinducible plant expression system that allows controllable, high levelexpression of foreign genes in plant hosts.

BACKGROUND OF THE INVENTION

Plants offer enormous potential for production of recombinant forms oftherapeutics and diagnostics for humans and animal applications, forexample, human blood proteins, antibodies, vaccines, and the like.Plants have the ability to perform complex post-translationalmodifications and are intrinsically safe since they do not propagatemammalian viruses or pathogens. Plants also offer a lower costalternative and are easier to scale-up compared to traditional mammaliancell culture production methods (Goldstein and Thomas, 2004; Ma et al.,2003). Thus, the potential for plant expression systems to serve aslarge scale production methods of therapeutic and diagnostic proteins,including the production of enzymes is compelling. Transient productionapproaches utilizing agroinfiltration or plant viral infection of planttissue are particularly promising for short production timelines due tothe relatively short time needed to go from gene to product, and theability to use available agricultural biomass for rapid, large scaleproduction (Fischer et al., 1999). Despite the ease and short turnaroundtime for transient production, use of recombinant plant viruses has beenhindered by various shortcomings, including low infection efficiency,the risks associated with the release of the competent recombinant virusvectors into the environment (Manske and Schiemann, 2005), limitationson the size of the transgene, and transgene instability (Rabindran andDawson, 2001; Shivprasad et al., 1999).

Agrobacterium-mediated infection to deliver viral amplicons was firstreported in 1993 (Turpen et al., 1993). Gleba et al. (2005) described aplant virus based expression technology, termed “magnifection”, whereinwhole plants are vacuum infiltrated with A. tumefaciens containing abinary vector capable of expressing recombinant viral RNA replicons ableto move from cell-to-cell, multiply, and express amplified levels oftransgenes carried by the viral replicon. Gils et al. have shown highlevel production of correctly processed, biologically active humangrowth hormone using transient agroinfiltration of N. benthamiana leaveswith multiple constructs that allow the in planta assembly of TMV viralreplicons (Gils et al., 2005). Production of heterologous proteinsutilizing constitutively-expressed viral amplicons has also beeninvestigated in stably transformed plants, however, low recombinantviral levels, and low product titers resulting from post-transcriptionalgene silencing (PTGS) have been continuously problematic (Angell andBaulcombe, 1997; Mori et al., 1993). In order to address this issue,Mori et al. (2001) constructed a chemically inducible Brome mosaic virus(BMV)-based amplicon utilizing the dexamethasone (DEX)glucocorticoid-inducible transcription system (Aoyama and Chua, 1997)for production of human gamma interferon in transgenic Nicotianabenthamiana. A similar approach was used for protein expression using aplant DNA virus amplicon (Zhang and Mason, 2006).

However, low recombinant viral levels, and low product titers resultingfrom post-transcriptional gene silencing continue to be a problem. Thus,with increasing threats of global pandemics and bioterrorism, there is acritical need for new bio-manufacturing technologies that allow rapid,large scale and cost-effective production of diagnostic, therapeutic andprophylactic compounds that can be used for detection, treatment orvaccination following such an event. The invention addresses this need.

There is also an increasing need for renewable and more environmentallyfriendly alternatives and supplements to fossil fuels and the efficient,low cost and scalable production of enzymes that are involved in thebiosynthesis of such fuels. While bioethanol and biodiesel are rapidlyexpanding as renewable, more environmentally friendly alternatives tofossil fuel derived gasoline and diesel, new technologies that allow,for example, energy efficient and cost efficient degradation oflignocellosic biomass would represent a major breakthrough. Althoughthere have been advances in improving the activity of enzymes involvedin the degradation of lignocellulosic biomass, design of engineeredcellulase mixtures, development of more productive host strains, andutilization of inexpensive medium components, the high cost ofproducing, recovering and formulating cellulase enzymes usingtraditional fungal or bacterial fermentation continues to impact theeconomics of ethanol production from lignocellulosic biomass. Microbialfermentations require large energy inputs due to agitation, aeration,temperature control and in some cases cell disruption and enzymerecovery, as well as high capital equipment costs for fermentors anddownstream processing unit operations. The application of cellulasesoften requires pretreatment of lignocellulosic biomass to facilitateaccessibility of the enzymes to the complex substrate, again requiringadditional energy inputs and/or costly treatment of acid/base wastes.Thus, a new approach for controlled, in planta production of enzymesinvolved in lignocellulosic degradation using chemically inducible,transient, high level expression of cellulase enzymes produced in plantsis highly desirable and the invention addresses this need.

SUMMARY OF THE INVENTION

The invention relates to a novel Cucumber mosaic virus (CMV) inducibleviral amplicon (CMViva) expression system that allows for tightlyregulated chemically-inducible expression of heterologous genes in planthosts. Transient production of recombinant α₁-antitrypsin (rAAT), ahuman blood protein, is shown in Nicotiana benthamiana leaves. Highproduction levels are obtained by co-infiltrating leaves withAgrobacterium tumefaciens cells containing CMViva carrying the AAT geneand A. tumefaciens cells carrying a binary vector constitutivelyexpressing the gene silencing suppressor p19 for example, from Tomatobushy stunt virus. Accumulation of up to thirty-fold more rAAT can beobserved in leaves (24 mg per 100 g leaf tissue) when compared with theexpression levels observed using the constitutive CaMV 35S promoter.Significantly, 70% of the rAAT produced using the CMViva expressionsystem is found to be biologically active, i.e., a 170-fold increase infunctional protein compared with the CaMV 35S expression system.

One aspect of the invention provides a plant expression system includinga cucumber mosaic virus inducible viral amplicon for the expression of aheterologous gene in a plant or plant cell. The expression system mayfurther include a cDNA coding for the cucumber mosaic virus which isoperably linked to the heterologous gene. In one embodiment, theheterologous gene codes for a human blood protein. The expression systemdisclosed herein may be present in a transgenic plant or transgenicplant cell suspension culture in which the expression cassette has beenstably incorporated into the plant genome and therefore inheritable byprogeny.

Another aspect of the invention provides a method for producing aheterologous protein. The method may include: a) contacting a livingplant, recently harvested tissue from a living plant, or plant cellswith a bacterial cell containing the expression system described herein;b) exposing the plant, excised plant tissue, or plant cells to thechemical inducer; c) growing and/or incubating the contacted plant,excised plant, or plant cells for a time period sufficient to producethe heterologous protein; and d) harvesting the heterologous proteinfrom the plant, excised plant tissue, plant cells or plant cellsuspension culture broth. In a further embodiment, the method mayinclude co-infiltrating the plant, excised plant tissue, or plant cellswith a bacterial cell that contains the expression system and abacterial cell that carries a binary vector constitutively expressing agene silencing suppressor. In another embodiment, the bacterial cellincludes Agrobacterium tumefaciens or the like. In yet anotherembodiment, the gene silencing suppressor is p19. In a furtherembodiment, the heterologous protein is a human protein. In yet afurther embodiment, the heterologous protein is a cell wall modifying ordegrading enzyme.

In one format, the invention relates to a cucumber mosaic virusinducible viral amplicon (CMViva) for the expression of heterologousgenes in plants. In another format, the CMViva expression system can bea single binary plasmid containing the heterologous gene replacing theCoat Protein gene of the cucumber mosaic virus. In another format, theCMViva expression system can include two binary plasmids, wherein thefirst plasmid contains the cDNA encoding the CMV1a and CMV2a replicasegenes, and the second plasmid contains the heterologous gene. The CMVivasystem is tightly regulated and inducible by the addition of estradiol.

One aspect of the invention provides the CMViva expression system thatis tightly regulated and chemically inducible by the addition of achemical such as, for example, mammalian steroid hormones such asestradiol. Regulation and induction of the system are conferred by theinsertion of cDNAs of the estradiol inducible expression system XVE anda LEX operator operably linked to the CMV1a replicase gene. The bindingof estradiol to the constitutively expressed XVE chimeric proteininitiates a translocation and binding of the dimerized XVE chimericprotein to the LEX operator, whereby the binding of the LEX operatorinitiates the expression of the CMV1a replicase gene. The expression ofthe CMV replicase gene subsequently initiates the expression process ofthe heterologous gene. In another embodiment, steroidal or nonsteroidalagonist of insect hormones can be substituted for mammalian steroidhormones. For example, the insect ecdysone receptor gene switch reportedby Tavva et al. 2006 could be used in place of the XVE/LEX operator.

Another aspect of the invention provides a method for producing aheterologous protein in a plant or plant cells by contacting the plantor plant cells with a bacterial cell, namely Agrobacterium tumefaciens,containing the expression system. The system may be stably integratedinto the plant genome or transiently expressed. After introduction ofthe bacterial cells and exposure to the chemical inducer, the plants orplant cells may be grown, cultured, or incubated for a sufficient timeperiod after which the heterologous protein is produced and harvested.Production of the heterologous protein is enhanced by introducing orco-infiltrating a plasmid containing the suppressor protein with theplasmid(s) containing expression system.

The heterologous gene can encode for other proteins, RNAs, or enzymes.

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS

The present invention is best understood when read in conjunction withthe accompanying tables and figures. It is understood, however, that theinvention is not limited to the specific embodiments disclosed in thetables or figures.

Table 1 depicts the list of primers used for amplification of DNA ofvarious genes by PCR.

Table 2 depicts the comparison of N. benthamiana growth and rAATproduction in bioreactors using different expression systems.

Table 3 depicts the comparison of recombinant AAT production usingCMViva system by transient expression in N. benthamiana leaves with andwithout co-infiltration with the p19 gene silencing suppressor, and thecomparison of transgenic N. benthamiana cell culture in a bioreactor(without p19 expression).

Table 4 depicts the comparison of recombinant human AAT production yieldand quality using CMViva system by transient expression in N.benthamiana leaves and transgenic N. benthamiana cell culture in abioreactor. a TSP=total soluble protein; b FW=Fresh tissue weight.

FIG. 1A depicts the CMViva expression system (modified from Mori; seeMori et al., 2001). This figure shows conceptually how the CMViva systemworks for the regulated, high level production of foreign proteins(e.g., AAT) in plant cells that have either been stably transformed oragroinfiltrated with A. tumefaciens containing the pCMV-SPAAT vector(see FIG. 2 below). In the absence of estradiol (E), only steps labeledwith I occur and no product (AAT protein) is produced. Once estradiol isadded, steps labeled with II occur as well as the steps labeled with I.First estradiol is taken up by the cells (step II 6); it binds to theconstitutively expressed XVE chimeric protein (step II 7) creating adimer (step II 8) that translocates to the nucleus and binds to the LexAoperator (step II 9) allowing transcription of the CMV RNA1 cDNA (stepII 10). The CMV RNA1 is transported out of the nucleus, and the ORF1A istranslated (step II 12) to generate CMV1a protein. Functional replicaseis produced (step II 13) from CMV1a, CMV2a and host factors (H).Functional replicase enables replication of RNA 2 and RNA 3 (steps II 14and II 15) as well as the generation of the subgenomic RNAs andtranscription of the AAT gene (step II 16) followed by translation toproduce the AAT protein product (step II 17). The CMV2b protein is alsoexpressed from subgenomic RNA only after step II 14, so it will not beproduced in the uninduced state. We have deleted 57 nucleotides on the5′ end of the CMV RNA 1 cDNA so that the positive sense CMV RNA 1acannot be replicated in the presence of functional replicase (as RNAs 2and 3 are), as would normally occur in wild type CMV. This RNA can onlyserve as an mRNA to generate the 1a protein that then allows activationof CMV replication. Thus, the supply of the key 1a protein is notautocatalytic but controlled directly by step II 10. As a result thisreplication-impaired system is less sensitive to leakiness, safer, andmore controllable.

FIG. 1B depicts linear maps of infectious clones pQCD1, pQCD2, andpQCD3, corresponding to CMV RNAs 1, 2, and 3, respectively, and binaryvectors pER10, pDU97.1005, and pDU99.2215, containing the promoters,genes, and transcription terminators used for the construction ofexpression vectors pCMV, pCMV-SPAAT, pXVE-SPAAT and p35S-SPAAT (see themain text and FIG. 2 below). Restriction sites used for the cloning areshown as A, Asc I; B, BamH I; Bg, Bgl II; E, EcoR I; H, Hind III; N, NotI; Ns, Nsi I; S, Sal I; Sp, Spe I; and X, Xho I. Primer positions anddirections are shown with a small arrow with primer names indicatedbelow the arrow (see Table 1).

^(a)pQCD2.1 was generated by linearizing pQCD2 with EcoR I and Spe I,filling with Klenow followed by self ligation.

^(b)pQCD2.2 was generated by digesting pQCD2.1 with Hind III and Bgl IIand ligating with pBluescript (Stratagene Inc, La Jolla, Calif.) cutwith Hind III and Bam HI.

^(c)PCR products generated using primer pairs O14G10Pf:O15NPeaTr andO16NOpf:O17XSOpr were cloned into pGEM-T (Promega Inc, Madison, Wis.),released as Sal I/Nsi I and Nsi EcoR I fragments, and ligated along withEcoR I/BamH I fragment from pQCD1 into pQCD2.2, cut with Sal I/BamH I,in a four fragment ligation.

^(d)PCR product generated using primer pair OQCD3-1:OQCD3-6 was digestedwith Xho I and Bgl II and ligated into pSP72 (Promega Inc.) cut with XhoI and Bgl II.

^(e)PCR products generated using primer pairs OQCD3-1: OQCD3-2,OQCD3-3:OQCD3-4, and OQCD3-5:OQCD3-6, were cloned using TOPO-Zerobluntcloning kit (Invitrogen, Carlsbad, Calif.) and the fragments werereleased as Xho EcoR I, EcoR I/BamH I and BamH Asc I fragments andligated with pQA cut with Xho I and Asc I in a four fragment ligation.

FIG. 2 depicts the map of constructs used in this study: (A) pCMV; (B)pCMV-SPAAT; (C) pXVE-SPAAT; and (D) p35S-SPAAT. LB, left border; RB,right border; P, promoter; T, terminator.

FIG. 3 depicts immunoblot analysis of CMV CP: Comparison between CMV CPproduction in N. benthamiana plants infiltrated with A. tumefacienscells containing pCMV. Lane 1: Control, CMV-infected Cucurbita pepoplants; lanes 2-4: pCMV+estradiol; lanes 5-7: pCMV-estradiol; lanes 8and 9: healthy control plant.

^(a)I=Plants treated with the estradiol inducer; (+) added; (−) notadded; (*) non applicable.

FIG. 4 depicts immunoblot analysis of total and functional rAAT.Comparison between rAAT productions in plants infiltrated with A.tumefaciens cells containing p35S-SPAAT, pXVE-SPAAT, or pCMV-SPAAT whenco-infiltrated with A. tumefaciens cells containing the silencingsuppressor p19. (A) Comparison of total rAAT: lane 1, MultiMarkmolecular weight marker (Invitrogen, Carlsbad, Calif.); lane 2, healthycontrol plant; lane 3, p35S-SPAAT; lanes 4 and 5, pXVE-SPAAT; lanes 6and 7, pCMV-SPAAT; lane 8, authentic human AAT (48 ng); lane 9 and 10,replicates of lane 4 and 6, respectively, using duplicate plants. (B)Band shift analysis for comparison of functional rAAT: lane 1, MultiMarkmolecular weight marker; lanes 2 and 3, p35S-SPAAT; lanes 4 and 5,pXVE-SPAAT; lanes 6 and 7, pCMV-SPAAT; lanes 8 and 9, human AAT standard(48 ng); lane 10, only PPE.

^(a)I=Plants treated with the estradiol inducer; ^(b)PPE=Supernatantsamples incubated with PPE before SDS-PAGE. (+) added; (−) not added;(*) non applicable.

FIG. 5 depicts immunoblot analysis of total and functional rAAT.Comparison between rAAT productions in plants infiltrated with A.tumefaciens cells containing pXVE-SPAAT or pCMV-SPAAT. (A) Comparison oftotal rAAT: lane 1, healthy control plant; lanes 2-5, pXVE-SPAAT; lanes6-9, pCMV-SPAAT; lane 10, human AAT standard (48 ng). (B) Band shiftanalysis for comparison of functional rAAT: lanes 1-4, pXVE-SPAAT; lanes5-8, pCMV-SPAAT; lanes 9 and 10, human AAT standard (48 ng).

^(a)I=Plants treated with the estradiol inducer; ^(b)PPE=Supernatantsamples incubated with PPE before SDS-PAGE; ^(c)p19=Plantsco-infiltrated with A. tumefaciens cells containing the p19 silencingsuppressor. (+) added; (−) not added; (*) non applicable.

FIG. 6 depicts ELISA analysis of transiently expressed total andfunctional rAAT. Data are the average results from triplicate plantsexposed to the same conditions at 2.5 days post-infiltration. Solidlines represent total rAAT and hatched lines represent functional rAAT.Error bars represent one standard deviation from triplicate assaysperformed on a given sample extract.

FIG. 7 depicts Western blotting and band shift analysis for comparisonof total and functional rAAT production by three expression systems.Lane 1, human AAT standard; lane 2, human AAT incubated with procinepancreatic elastase (PPE); lane 3, Multimark molecular marker; lane 4,pCMV-spAAT (without induction); lane 5, pCMV-spAAT (with induction);lane 6, pCMV-spAAT (with induction) incubated with PPE; lane 7,pXVE-spAAT (without induction); lane 8, pXVE-spAAT (with induction);lane 9, pXVE-spAAT (with induction) incubated with PPE; lane 10, wildtype N. benthamiana; lane 11, p35S-spAAT; lane 12, p35S-spAAT incubatedwith PPE. A band at 52 kDa corresponds to human AAT and a band at 48 kDacorresponds to rAAT. In the band shift assay, a band at a molecularweight around 75-80 kDa represent the human AAT-PPE covalent complex anda lower band is the cleaved product of AAT-PPE reaction.

aI=Plant cell culture treated with the estradiol inducer; bPPE=Supernatant samples incubated with PPE before SDS-PAGE; (+) added;(−) not added; (*) non applicable.

FIG. 8 depicts the effect of concentration of inducer (estradiol) onextracellular total and functional rAAT production from the XVE andCMViva inducible cell cultures. Inducer solution was applied toinducible cell cultures (10 days old, 100 mL in a 250 mL flask) at afinal concentration of 0, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50 or 100 μMestradiol. Samples were taken at day 8 after starting induction phasefor AAT ELISA analysis. Error bars represent one standard deviation ofmeasurements obtained from duplicate experiments.

FIG. 9 depicts the effect of timing of induction on extracellular totaland functional rAAT production in the XVE inducible cell cultures.Inducer solution was added into XVE cell culture (100 mL culture in a250 mL flask) to a final concentration of 10 μM estradiol at day 5, 8,11 or 14 after inoculation. Samples were analyzed by AAT ELISA. Dashedlines represent the time of addition of inducer at day 5, 8, 11 or 14after inoculation.

FIG. 10 depicts the effect of timing of induction on extracellular totaland functional rAAT production in the CMViva inducible cell cultures.Inducer solution was added into CMViva cell culture (100 mL culture in a250 mL flask) to a final concentration of 10 μM estradiol at day 5, 8,11 or 14 after inoculation. Samples were analyzed by AAT ELISA. Dashedlines represent the time of addition of inducer at day 5, 8, 11 or 14after inoculation.

FIG. 11 depicts the growth kinetic comparisons of transgenic plant cellcultures with 35S, XVE or CMViva expression systems and wild type N.benthamiana in a bioreactor. Dashed lines represent the time of additionof inducer at day 11 after inoculation for the XVE and CMViva systems.

FIG. 12 depicts the comparisons of rAAT production profiles fortransgenic plant cell cultures with 35S, XVE or CMViva expressionsystems and wild type N benthamiana in a bioreactor. Dashed linesrepresent the timing of induction at day 11 after inoculation for theXVE and CMViva systems.

FIG. 13 depicts the kinetics of biomass accumulation and extracellularestradiol concentration in wild type N. benthamiana cell cultures grownin a flask. The dashed line represents the timing of induction at day 10after inoculation.

FIG. 14 depicts the Western blotting analysis of the stability anddegradation of human AAT in cell-free conditioned medium prepared from 7day old wild type N. benthamiana or CMViva transgenic plant cellcultures. Day represents the time after incubation. Functional AAT (%)indicates the relative amount of functional human AAT remaining as apercentage of the initial concentration determined by functional AATELISA.

FIG. 15 depicts the kinetic profiling of the stability and degradationof human AAT in cell-free conditioned medium prepared from 7 day oldCMViva transgenic plant cell cultures. Day represents the time afterincubation. Relative total and functional human AAT (%) indicates therelative amount of total and functional human AAT remaining as apercentage of the initial concentration determined by AAT ELISA.hAAT/fresh, sterilized KCMS: human AAT incubated in fresh and filtersterilized KCMS medium; hAAT/CMViva day 7, pH 5.1: human AAT incubatedin cell-free 7 day old CMViva cell cultured medium with pH 5.1;hAAT/CMViva day 7, pH 6.4: human AAT incubated in cell-free 7 day oldCMViva cell cultured medium with changed pH 6.4; hAAT/CMViva day 7,heated: human AAT incubated in cell-free 7 day old CMViva cell culturedmedium that was heated at boiling water for 30 min prior to the additionof human AAT; hAAT/CMViva day 7+2-Mer: human AAT incubated in cell-free7 day old CMViva cell cultured medium containing2-mercaptoethylamine-HCl (aminopeptidase inhibitor); hAAT/CMViva day7+AEBSF: human AAT incubated in cell-free 7 day old CMViva cell culturedmedium containing AEBSF (broad spectrum serine and cysteine proteasesinhibitor); hAAT/CMViva day 7+Benzamidine: human AAT incubated incell-free 7 day old CMViva cell cultured medium containingbenzamidine-HCl (serine protease inhibitor); hAAT/CMViva day 7+EDTA:human AAT incubated in cell-free 7 day old CMViva cell cultured mediumcontaining EDTA (a metal ion chelator, anti-metalloproteinase). Thefinal working concentration for each protease inhibitor is 10 mM. Errorbars represent one standard deviation of measurements obtained fromduplicate experiments.

FIG. 16A depicts the Western blotting analysis of human AAT incubated invarious cell-free conditioned medium prepared from 7 day old CMVivatransgenic plant cell cultures. Samples were taken on 1 day afterincubation. Lane 1, human AAT standard; lane 2, human AAT incubated infresh and sterilized KCMS medium; lane 3, human AAT incubated incell-free conditioned medium with changed pH 6.4; lane 4, human AATincubated in cell-free conditioned medium that was heated at boilingwater for 30 min prior to the addition of human AAT; lane 5, human AATincubated in cell-free conditioned medium containing2-mercaptoethylamine-HCl; lane 6, human AAT incubated in cell-freeconditioned medium containing AEBSF; lane 7, human AAT incubated incell-free conditioned medium containing benzamidine-HCl; lane 8, humanAAT incubated in cell-free conditioned medium containing EDTA; lane 9,human AAT incubated in cell-free conditioned medium with original pH5.1; lane 10, multimark molecular marker.

FIG. 16B depicts band shift analysis of the human AAT incubated invarious cell-free conditioned medium prepared from 7 day old CMVivatransgenic plant cell cultures. Samples were taken on 1 day afterincubation. The layout of lanes is the same as FIG. 16A.

FIG. 17 depicts kinetics of functional human AAT protein stability incell-free conditioned medium collected from different ages of CMVivacell cultures including 7 days (medium pH 5.1), 11 days (medium pH 5.9)or 16 days (medium pH 6.9) old post-subculture. Error bars represent onestandard deviation of measurements obtained from duplicate experiments.

FIG. 18 depicts the degradation profile of functional human AAT in 11days old conditioned CMViva cell culture medium (free of cell) that wasregulated to different medium pH (4, 5, 6, 7 or 8). Error bars representone standard deviation of measurements obtained from duplicateexperiments.

FIG. 19 depicts the effects of medium exchange and pH control on rAATproduction in CMViva cell cultures in flask. Arrow indicates the timingof induction at day 10 after inoculation. Error bars represent onestandard deviation of measurements from duplicate experiments.

FIG. 20 depicts the effects of pH control on cell growth and rAATproduction in XVE cell cultures in a bioreactor. Dashed lines representthe timing of induction at day 11 after inoculation.

FIG. 21 depicts the effects of pH control on cell growth and rAATproduction in CMViva cell cultures in a bioreactor. Dashed linesrepresent the timing of induction at day 11 after inoculation.

FIG. 22 depicts the effects of pH control on cell growth and rAATproduction in 35S cell cultures in a bioreactor. Dashed lines representthe pH control timing at day 12 or 16 after inoculation.

FIG. 23 depicts the Bradford analysis of TSP in samples from N.benthamiana leaves exposed to various induction treatment methods:either no induction, a single topical application, multiple topicalapplications, a single pressure injection, or multiple pressureinjections. Data are grouped by method of inducer application and arethe average results from triplicate (for intact) of quadruplicate (fordetached) plant leaves exposed to the same conditions. Solid-filledlines represent production levels in intact leaves and hatched linesrepresent production levels in detached leaves. Error bars represent onestandard deviation from triplicate assays performed on samples oftriplicate or quadruplicate plant leaves.

FIG. 24 depicts the ELISA analysis of transiently expressed total rAATin samples from N. benthamiana leaves exposed to various inductiontreatment methods: either no induction, a single topical application,multiple topical applications, a single pressure injection, or multiplepressure injections. Data are grouped by method of inducer applicationand are the average results from triplicate (for intact) ofquadruplicate (for detached) plant leaves exposed to the sameconditions. Solid-filled lines represent production levels in intactleaves and hatched lines represent production levels in detached leaves.Error bars represent one standard deviation from triplicate assaysperformed on samples of triplicate or quadruplicate plant leaves.

FIG. 25 depicts the ELISA analysis of transiently expressed total rAATin samples from N. benthamiana leaves exposed to various inductiontreatment methods: either no induction, a single topical application,multiple topical applications, a single pressure injection, or multiplepressure injections. Data are grouped by method of inducer applicationand are the average results from triplicate (for intact) ofquadruplicate (for detached) plant leaves exposed to the sameconditions. Solid-filled lines represent production levels in intactleaves and hatched lines represent production levels in detached leaves.Error bars represent one standard deviation from triplicate assaysperformed on samples of triplicate or quadruplicate plant leaves.

FIG. 26 depicts the ELISA analysis of transiently expressed biologicallyfunctional rAAT in samples from N. benthamiana leaves exposed to variousinduction treatment methods: either no induction, a single topicalapplication, multiple topical applications, a single pressure injection,or multiple pressure injections. Data are grouped by method of inducerapplication and are the average results from triplicate (for intact) ofquadruplicate (for detached) plant leaves exposed to the sameconditions. Solid-filled lines represent production levels in intactleaves and hatched lines represent production levels in detached leaves.Error bars represent one standard deviation from triplicate assaysperformed on samples of triplicate or quadruplicate plant leaves.

FIG. 27 depicts the first of the two binary plasmids of in thealternative construction of the CMViva expression system (Example 2).

FIG. 28 depicts the second of the two binary plasmids of in thealternative construction of the CMViva expression system (Example 2).

DETAILED DESCRIPTION OF THE INVENTION

a.) A Chemically Inducible Plant Viral Amplicon Expression System

The invention relates to a novel chemically inducible plant viralamplicon (CMViva) expression system that permits controllable, highlevel expression of foreign genes in plant hosts. This system can beused in transient agro-infiltration in intact, living plants, recentlyharvested plant tissues (e.g. leaves, stems), or plant cells, and alsofor stable integration into the genomes of plants or plant cells toallow production of a protein, enzyme or RNA of interest. This systemprovides a major advantage over existing plant expression systemsbecause it allows for consistent expression of foreign or heterologousproteins in plant hosts. The invention encompasses geneticallyengineered plant cells that contain modified complementary DNAs (cDNAs)representing the complete tripartite genome of Cucumber mosaic virus(CMV), in which the CMV coat protein gene has been replaced by a targetgene of interest, which along with other modifications ensure that noinfectious CMV virus is generated. In this system, one of the keyCMV-encoded protein components of the viral replicase is under thecontrol of a relatively tightly regulated chemically-inducible promoter.As such, the recombinant viral amplicons are only producedintracellularly and under specific induction conditions (FIG. 1A).

One aspect of the invention provides the CMViva expression system thatis tightly regulated and chemically inducible by the addition of achemical such as, for example, mammalian steroid hormones such asestradiol. When estradiol is the chemical inducer, regulation andinduction is achieved by the insertion of cDNAs of the estradiolinducible expression system XVE and a LEX operator. The binding ofestradiol to the constitutively expressed XVE chimeric protein initiatesa translocation and binding of the dimerized XVE chimeric protein to theLEX operator, whereby the binding of the LEX operator initiates theexpression of the CMV replicase gene. The expression of the CMVreplicase gene subsequently initiates the expression process of theheterologous gene.

In a further embodiment, steroidal or nonsteroidal agonist of insecthormones can be substituted for mammalian steroid hormones. For example,the insect ecdysone receptor (EcR) gene switch reported by Tavva et al.2006 could be used in place of the XVE/LEX operator to create aEcR-based inducible gene expression system. In this system,methoxyfenozide is the chemical inducer, whereby the presence ofmethoxyfenozide induces the expression a heterologous gene.

Another aspect of the invention provides the CMViva expression systemoperably linked to a gene of interest (e.g., a bacterial, plant, yeast,fungi, or animal). In one embodiment, a plant (e.g., leaf, stem, etc.)is infiltrated with bacterial (Agrobacterium tumefaciens) cells thatcontain the CMViva expression system operably linked to a gene ofinterest (e.g., a human gene that codes for a human protein). In anotherembodiment, a plant (e.g., leaf, stem, etc.) is infiltrated withbacterial cells that contain the CMViva expression system operablylinked to a gene of interest (e.g., a human gene that codes for a humanprotein) and bacterial cells that carry a binary vector thatconstitutively expresses the gene silencing suppressor p19, the genesilencing suppressor p19 can be from Tomato bushy stunt virus. In afurther embodiment, the CMViva is stably integrated into a plant orplant cells of interest.

b.) Definitions

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced. The disclosures of thesepublications, patents and published patent specifications are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques of plant breeding, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook, Fritsch andManiatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989);CURRENT PROTOCOLS IN MOLECULAR BIOLOGY [(F. M. Ausubel, et al. eds.,(1987)]; PLANT BREEDING: PRINCIPLES AND PROSPECTS (Plant Breeding,Vol. 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall,(1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995)CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc.); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)],Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE [R. I. Freshney, ed. (1987)].

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes V, published by Oxford University Press, 1994(SBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (SBN0-632-02182-9); and Robert A. Meyers (ed), Molecular Biology andBiotechnology, a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Ausubel et al. (1987)Current Protocols in Molecular Biology, Green Publishing; Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.

In order to facilitate review of the various embodiments of theinvention, the following definitions are provided:

-   Non-naturally Occurring Plant: A non-naturally occurring plant is a    plant that does not occur in nature without human intervention.-   Sequence Identity The similarity between two nucleic acid sequences    or two amino acid sequences is expressed in terms of sequence    identity (or, for proteins, also in terms of sequence similarity).    Sequence identity is frequently measured in terms of percentage    identity; the higher the percentage, the more similar the two    sequences are. Homologs and variants of the nucleic acid molecules    described herein may be used in the present invention. Homologs and    variants of these nucleic acid molecules will possess a relatively    high degree of sequence identity when aligned using standard    methods. Such homologs and variants will hybridize under high    stringency conditions to one another. Methods of alignment of    sequences for comparison are well known in the art. Various programs    and alignment algorithms are described in: Smith and Waterman    (1981); Needleman and Wunsch (1970); Pearson and Lipman (1988);    Higgins and Sharp (1988); Higgins and Sharp (1989); Corpet et al.    (1988); Huang et al. (1992); and Pearson et al. (1994). Altschul et    al. (1994) presents a detailed consideration of sequence alignment    methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. It can be accessed at the NCBIWebsite. A description of how to determine sequence identity using thisprogram is available at the NCBI website.

Homologs of the disclosed protein and nucleic acid sequences aretypically characterized by possession of at least 40% sequence identitycounted over the full length alignment with the amino acid sequence ofthe disclosed sequence using the NCBI Blast 2.0, gapped blastp set todefault parameters. The adjustable parameters are preferably set withthe following values: overlap span 1, overlap fraction=0.125, wordthreshold (T)=11. The HSP S and HSP S2 parameters are dynamic values andare established by the program itself depending upon the composition ofthe particular sequence and composition of the particular databaseagainst which the sequence of interest is being searched; however, thevalues may be adjusted to increase sensitivity. Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at leastabout 50%, at least about 60%, at least about 70%, at least about 75%,at least about 80%, at least about 90% or at least about 95% sequenceidentity.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequences in thefigures, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than that shown in thefigures as discussed below, will be determined using the number of aminoacids in the longer sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedherein for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion. As will be appreciated by those skilled in the art, thesequences of the present invention may contain sequencing errors. Thatis, there may be incorrect nucleotides, frameshifts, unknownnucleotides, or other types of sequencing errors in any of thesequences; however, the correct sequences will fall within the homologyand stringency definitions herein.

-   High Stringency High stringency conditions refers to hybridization    to filter-bound DNA in 5×SSC, 2% sodium dodecyl sulfate (SDS), 100    ug/ml single stranded DNA at 55-65° C., and washing in 0.1×SSC and    0.1% SDS at 60-65° C.-   Vector: A nucleic acid molecule as introduced into a host cell,    thereby producing a transformed host cell. A vector may include one    or more nucleic acid sequences that permit it to replicate in one or    more host cells, such as origin(s) of replication. A vector may also    include one or more selectable marker genes and other genetic    elements known in the art.-   Transformed: A transformed cell is a cell into which has been    introduced a nucleic acid molecule by molecular biology techniques.    As used herein, the term transformation encompasses all techniques    by which a nucleic acid molecule might be introduced into such a    cell, plant or animal cell, including transfection with viral    vectors, transformation by Agrobacterium, with plasmid vectors, and    introduction of naked DNA by electroporation, lipofection, and    particle gun acceleration and includes transient as well as stable    transformants.-   Isolated: An “isolated” biological component (such as a nucleic acid    or protein or organelle) has been substantially separated or    purified away from other biological components in the cell or the    organism in which the component naturally occurs, i.e., other    chromosomal and extra chromosomal DNA and RNA, proteins and    organelles. Nucleic acids and proteins that have been “isolated”    include nucleic acids and proteins purified by standard purification    methods. The term embraces nucleic acids including chemically    synthesized nucleic acids and also embraces proteins prepared by    recombinant expression in vitro or in a host cell and recombinant    nucleic acids as defined below.-   Operably linked: A first nucleic acid sequence is operably linked    with a second nucleic acid sequence when the first nucleic acid    sequence is placed in a functional relationship with the second    nucleic acid sequence. For instance, a promoter is operably linked    to a protein coding sequence if the promoter affects the    transcription or expression of the protein coding sequence.    Generally, operably linked DNA sequences are contiguous and, where    necessary, join two protein coding regions in the same reading    frame. With respect to polypeptides, two polypeptide sequences may    be operably linked by covalent linkage, such as through peptide    bonds or disulfide bonds.-   Recombinant: By “recombinant nucleic acid” herein is meant a nucleic    acid that has a sequence that is not naturally occurring or has a    sequence that is made by an artificial combination of two otherwise    separated segments of sequence. This artificial combination is often    accomplished by chemical synthesis or, more commonly, by the    artificial manipulation of nucleic acids, e.g., by genetic    engineering techniques, such as by the manipulation of at least one    nucleic acid by a restriction enzyme, ligase, recombinase, and/or a    polymerase. Once introduced into a host cell, a recombinant nucleic    acid is replicated by the host cell, however, the recombinant    nucleic acid once replicated in the cell remains a recombinant    nucleic acid for purposes of this invention. By “recombinant    protein” herein is meant a protein produced by a method employing a    recombinant nucleic acid. As outlined above “recombinant nucleic    acids” and “recombinant proteins” also are “isolated” as described    above.-   Ortholog: Two nucleotide or amino acid sequences are orthologs of    each other if they share a common ancestral sequence and diverged    when a species carrying that ancestral sequence split into two    species, sub species, or cultivars. Orthologous sequences are also    homologous sequences. Orthologous sequences hybridize to one another    under high-stringency conditions. The term “polynucleotide”,    “oligonucleotide”, or “nucleic acid” refers to a polymeric form of    nucleotides of any length, either deoxyribonucleotides or    ribonucleotides, or analogs thereof. The terms “polynucleotide” and    “nucleotide” as used herein are used interchangeably.    Polynucleotides may have any three-dimensional structure, and may    perform any function, known or unknown. The following are    non-limiting examples of polynucleotides: a gene or gene fragment,    exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,    ribozymes, cDNA, recombinant polynucleotides, branched    polynucleotides, plasmids, vectors, isolated DNA of any sequence,    isolated RNA of any sequence, nucleic acid probes, and primers. A    polynucleotide may comprise modified nucleotides, such as methylated    nucleotides and nucleotide analogs. If present, modifications to the    nucleotide structure may be imparted before or after assembly of the    polymer. The sequence of nucleotides may be interrupted by    non-nucleotide components. A polynucleotide may be further modified    after polymerization, such as by conjugation with a labeling    component. A “fragment” or “segment” of a nucleic acid is a small    piece of that nucleic acid.

c.) Uses of the Non-naturally Occurring Plants of the Invention

The non-naturally occurring plants of the invention are specificallyuseful in that they can be employed as hosts for production of foreignor heterologous proteins. As such, the non-naturally occurring plantscan be used to provide various amounts of foreign protein content,including therapeutic proteins that can be used as drugs, diagnosticproteins that can be used as markers, and the like. Examples of aforeign protein that can be produced in a plant are cellulases forcontrolled in-planta degradation of lignocellulose for use as afeedstock for ethanol production and others. As defined herein, foreignor heterologous protein content means that the plant produces a proteinthat does not occur within the natural plant. The non-naturallyoccurring plants of this invention employ the CMViva expression systemthat allows for tightly regulated chemically-inducible expression ofprotein coding genes in the non-naturally occurring plants. In oneembodiment, a plant (e.g., leaf, stem, etc.) is infiltrated withbacterial or Agrobacterium tumefaciens cells that contain the CMVivaexpression system operably linked to a gene of interest (e.g., a humangene that codes for a human protein). In another embodiment, a plant(e.g., leaf, stem, etc.) is infiltrated with bacterial or Agrobacteriumtumefaciens cells that contain the CMViva expression system operablylinked to a gene of interest (e.g., a human gene that codes for a humanprotein) and bacterial or Agrobacterium tumefaciens cells that carry abinary vector that constitutively expresses the gene silencingsuppressor p19.

The protein coding sequence linked to the CMViva expression system maybe any heterologous protein that is useful in optimizing plants forbiofuel production, therapeutics, vaccines, diagnostics or the like.Heterologous proteins useful in the invention include proteins encodedby polynucleotides from any source, natural or synthetic. Suitablecoding regions encode animal RNAs or polypeptides, as well as variants,fragments and derivatives thereof. The encoded products may be recoveredfor use outside the host plant cell (e.g., therapeutically activeproducts) or utilized in-planta for modification of the plant biomass(e.g., cellulase production). Examples of such coding regions includepolynucleotides derived from vertebrates, such as coding regions forRNAs (e.g., anti-sense RNAs, ribozymes, and chimeric RNAs havingribozyme structure and activity) or polypeptides (e.g. polypeptidecoding regions). Other coding regions useful in the inventive methodsare derived from other life forms such as yeast, fungi and bacteria. Theheterologous proteins which find particular use in the invention includethose that provide a therapeutic and/or diagnostic use in human andother animals. Such protein sequences are available in the literatureand known to those of skill in the art.

d.) In planta Production of Specific Enzymes Via the CMViva ExpressionSystem

The CMViva expression system can be used to express various proteins ofinterest including enzymes involved catalyzing new biofuels asenvironmentally friendly alternatives. Such enzymes include enzymesinvolved in lignocellulosic degradation (e.g., low cost, scalable andenergy efficient degradation of lignocellosic biomass) and enzymes usedfor oil bioconversion (e.g., conversion of feedstock triglycerides andfree fatty acid).

A. In planta Production of Enzymes Involved in LignocellulosicDegradation

The invention provides a new system for controlled, in planta productionof enzymes involved in lignocellulosic degradation using chemicallyinducible, transient, high level expression of cellulase enzymesproduced in plants. There are several potential applications of thistechnology:

-   1. The rapid, high level expression of functional cellulase enzymes    in the plant tissue just prior to harvesting (or just after    harvesting the biomass), results in the degradation of    lignocellulose, breakdown of plant tissues and generation of sugars.    A “mash” of the plant biomass may be directly used as a feedstock    for ethanol fermentation.-   2. The system can be used as a screening platform for rapid    production and evaluation of alternative and/or engineered cellulase    enzymes and mixtures for different plants/substrates. One of the    advantages of in planta screening is that plant tissues are    comprised of the heterogenous, natural lignocellulosic material    rather than an idealized laboratory substrate.-   3. The system can be used for large scale production of enzymes to    be used for degradation of nonviable or dried plant biomass (straws,    woods, etc.), similarly to microbially derived enzymes but at a    lower cost and reduced energy input.

One aspect of the invention provides expression systems for transientand stable production of recombinant proteins in plants and plant cellcultures. These can be used for rapid screening and production ofcellulase enzymes in plant tissues. One embodiment of the inventionprovides for agroinfiltration of nontransgenic plant tissues withrecombinant Agrobacterium tumefaciens containing the expression cassettefor inducible, transient expression of cellulases. The inventionencompasses chemically inducible expression systems such as the Cucumbermosaic virus inducible viral amplicon (CMViva) expression system. Thesystem can be used in plant leaves, including Nicotiana benthamianaplant leaves for production of a heterologous protein. The CMViva systemallows for the production of the recombinant protein upon theapplication of the chemical inducer, estradiol, to the plant leaf. Inorder to obtain a higher level and more controlled expression, theCucumber mosaic virus (CMV) is engineered into the CMViva expressionssystem. CMV is a plant virus which has a wide host range among dicot andmonocot plants, such that genes of interest can be inserted in place ofthe CMV gene which would normally encode the CMV capsid protein. Theentire CMV genome is delivered to plant cells by agro-infiltration,transformation, or stable integration, and CMV replication and productproduction is subsequently induced by adding estradiol. During CMVreplication the gene of interest is expressed at high levels due to geneamplification by CMV. As an added precaution, the CMV is engineered sothat it does not move within plants beyond the point of infiltration,and so that it cannot move between plants. In one embodiment, asecretion signal peptide is used to target the recombinant protein tothe apoplast. When the CMV coat protein replaced by AAT containing asecretion signal peptide, the presence of extracellular AAT can beverified in the transgenic CMViva plant cell suspension culturesfollowing the addition of estradiol to the medium. Thus, the cellulasesproduced within the plant cells can be to the apoplast where they have ahigh degree of accessibility to the cell wall matrix which enhancestheir effectiveness.

The process for transient agroinfiltration is known in the art (seeSudarshana et al. (2006) Plant Biotechnology Journal, 4:551-559). Asolution containing the recombinant A. tumefaciens can be applied to theleaf tissue through pressure injection using a sterile syringe (withouta needle), and the estradiol solution is applied to the agroinfiltratedarea about 12 hours later to induce expression. Maximum expressionlevels of recombinant protein (over 1% total soluble protein offunctional recombinant AAT, a particularly unstable protein, wasobtained) are observed about 2 day after induction. Some of theadvantages of this method are:

-   -   There is no need to generate or use transgenic plants.    -   The co-infiltration with multiple recombinant A. tumefaciens can        be used for simultaneous expression of different enzymes.    -   There is a short production time—about 4 days total from frozen        agro stock to protein production.    -   There are minimal energy requirements (only for agrobacterial        fermentation and plant tissue harvesting/processing).    -   A variety of plant species can be used with the expression        system.    -   The systems allows for the rapid and easy screening of different        enzymes by merely inserting the desired gene into the CMViva        expression cassette and performing agroinfiltration on        non-transgenic plants.

The invention further provides agroinfiltration processes that can beapplied to freshly harvested plant tissues (see McDonald andVanderGheynst (2005) Biotechnology Progress 22:723-730) which providesadvantages with respect to scale-up of the agroinfiltration andinduction processes. For example, see FIGS. 23-26.

The process of stable transformation of plants using agrobacterium iswell established. One aspect of the invention provides an inducibleplant based expression system for the in planta production of known andnovel cellulase genes. Plant-associated microbes, including endophytes,mutualists and a number of plant pathogens, encode for enzymes thatallow the respective microbes to obtain nutrients from the associatedplant, and as such are optimal sources of genes of interest. Mostendophytes and mutualists survive in concert with the plant andtherefore secrete enzymes that allow the microbe to obtain nutrients butdo not disrupt plant health or fitness. In contrast, pathogens, bydefinition, cause disease in plants, but they also encode for enzymesthat allow them to utilize plant components including lignin, cellulose,and pectin, as nutritional substrates. However, not all plant pathogenscause diseases that are similar in severity. Biotrophic plant pathogensare more generally dependent on a living host, and although they obtainnutrients by digesting plant parts, they regulate the quality andquantity of specific enzymes and do not generally destructively degradeand kill the host cell. By contrast, nectrotrophic plant pathogens aremore “brute force” and often secrete greater quantity or quality ofenzymes that can result in complete maceration and destruction of planthost tissues. Thus, plant-associated microbes offer a diverse andabundant source of enzymes that can be used to degrade plant componentsincluding cellulose. Due to rapidly accumulating genome information, thedesired genes can be identified from GenBank and other public databases,can easily be isolated using standard PCR approaches, can be cloned (andcodon optimized if desired), can be chemically synthesized and insertedinto the CMViva expression cassettes for expression in plants. In oneembodiment, the system can rapidly express different enzymes in plantsvia agroinfiltration. The proteins can be recovered from the infiltratedtissues and tested for activity on the desired substrates. This allowsfor rapid identification of useful enzymes, which can subsequently beproduced in whole plants and if desired, then scaled-up to production inbioreactors.

B. In Planta Production of Enzymes Involved in Oil Bioconversion

The invention provides a new system for controlled, in planta productionof enzymes involved as biocatalysts and/or chemical reaction steps forefficient production of bulk jet fuel surrogate and/or fuel additivesfrom plant, yeast or algal oils (e.g., triglycerides). Such enzymes areinvolved, for example, into conversion of feedstock triglycerides andfree fatty acids. Use of enzymes rather than chemical, thermal orinorganic catalytic methods has a number of advantages including higherspecificity, milder conditions, lower energy input requirements, andreduced waste and minimal environmental impact since enzymes arebiodegradable. For example, either chemical (acid-catalyzed oralkali-catalyzed) or enzymatic (lipase-catalyzed) approaches can be usedfor the transesterification of triglycerides to produce fatty acidmethyl esters FAME (known as biodiesel). Use of lipase catalyzedtransesterification has a number of advantages over alkali-catalyzedtransesterification, including lower energy requirements (the lipaseprocess reaction temperature is 30-40° C. while the alkali process is60-70° C.); easier recovery of free glycerol; insensitivity to watercontent in feedstock oil (for the alkali-catalyzed process the oils mustbe anhydrous because water results in saponification, producing soap,which reduces catalytic efficiency and alters the physical properties ofthe product); the ability to convert free fatty acids in the feedstockto FAME; and no more need for alkali wastewater treatment. A widevariety of lipases have been identified, characterized, produced inrecombinant systems, and produced commercially for a variety ofindustrial applications. With the advent of genetic engineeringapproaches it is possible to tailor an enzyme's catalytic abilitythrough approaches such as directed molecular evolution, thereby openingup new routes for enzyme engineering in order to enhance specificity,stability and catalytic efficiency under process conditions. The maindrawback to the biocatalytic route is the high cost associated with themanufacturing and purification of the enzymes, including the difficultyin re-using them due to their inherent instability. The invention offersa novel and much less costly approach, namely to produce the requiredenzymes directly in plant leaves via the CMViva expression system.

Examples of lipases which break down storage triglycerides into fattyacids are fatty acyl-CoA reductase (FAR) which converts fatty acids tofatty aldehydes and fatty aldehyde decarbonlyase (FAD) which convertsthe fatty aldehyde to a shorter (one carbon less) alkane/alkene andliberates carbon monoxide. Many lipases have been identified,characterized, produced in recombinant systems, and producedcommercially for a variety of industrial applications (see Hasan et al.,2006).

Once the target enzyme for the bioconversion process has beenidentified, the enzyme can be expressed using the procedures describedherein and produced by procedures well know in the art.

EXAMPLES

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

Example 1 Construction of the CMViva Expression System 1

The following materials were used in this example:

The complete cDNA clones, pQCD1, pQCD2, and pQCD3, corresponding togenomic RNAs 1, 2 and 3, of CMV strain Q (Ding et al., 1995),respectively.

The binary plasmid vectors pER8 (Zuo et al., 2000) and pER10 containingthe estradiol inducible expression system (XVE system); plasmid pER10 issimilar to pER8 except it contains nptII as the selectable markerinstead of the hygromycin resistance gene.

The binary vector p35S:p19 (Voinnet et al., 2003) containing the TBSVp19 gene

The binary vectors pDU97.1005 (Uratsu and Dandekar, unpublished), amodified version of pCGN1547 (McBride and Summerfelt, 1990)), pDU99.2215(Escobar et al., 2001) and pART7 (Gleave, 1992).

A. tumefaciens strain EHA105:pCH32 (Hamilton, 1997) was used to carryall the binary vectors used in this study except for p35S:p19, where A.tumefaciens strain C58C1 was used.

A series of binary plasmids were constructed to give inducible proteinexpression in plants after agroinfiltration (FIG. 2). The plasmid pCMVwas constructed by inserting a Hind III/Not I fragment from pQCD2.2, aNot I fragment from pXLRN1, and an Asc I fragment from pQA,sequentially, into pDU97.1005 (see FIG. 1B and Table 1 below for linearmaps of parental and intermediate plasmids and primers used). ForpCMV-SPAAT, a plant codon-optimized gene encoding for the AAT genecontaining a signal peptide (SP) sequence from rice α-amylase (SPAAT)(courtesy of Ventria Biosciences, West Sacramento, Calif.) was amplifiedusing primers OSDAATPst and OSDAATCPst, digested with Pst I and ligatedwith pQA3 linearized with Pst I. From the resultant clone an Asc Ifragment was used to construct pCMV-SPAAT as described for pCMV. Theplasmid pCMV contained all three CMV genomic components. The regionscoding for CMV RNAs 2 and 3 were controlled by the Cauliflower mosaicvirus (CaMV) 35S promoter as in the original plasmids, (pQCD2 and pQCD3)but the gene for CMV RNA 1 was modified to be under control of theestradiol-activated LEX operator. The plasmid pCMV-SPAAT was similar topCMV, except that the CMV coat protein (CP) gene (on the RNA 3 segment)was replaced with SPAAT. The plasmid pXVE-SPAAT was constructed byinserting the SPAAT sequence into pER10 so that SPAAT was under controlof the LEX operator. Finally, p35S-SPAAT was constructed by insertingSPAAT next to 35S promoter in pART7 and transferring a Not I fragmentinto pDU99.2215.

Example 2 Construction of the CMViva Expression System 2

Whereas the CMViva expression system in Example 1 is a binary pCMV-SPAATplasmid (FIG. 2B), CMViva expression system can also be two separatebinary plasmids. The first binary plasmid is the pDUR22XLR1R (FIG. 27);the second binary plasmid is comprised of a series of ten plasmidscontaining the AscI fragment for pQA (FIG. 28) cloned into pDU97 givingplasmids pQA-2, pQA-4, pQA-6, pQA-7, pQA-8, pQA-9, pQA-10, pQA-11,pQA-12, and pQA-13, collectively called the pQA series hereafter. ThepQA series is modified to have restriction sites including, but notlimited to, Eco RI-Sac, I-Kpn, 1-Sma, I-BamHI-Xba, I-Acc, I-Sal, I-Pst,and I-SphI-Hind III. The heterologous gene is cloned into the multiplecloning site (MCS) of one or all of the pQA series of plasmids. Thedifferent restriction sites used for cloning the recombinant genes allowfor easier and more efficient cloning and expression of the heterologousgenes, as well as for the identification of optimal sequences forprotein expression.

The first and second binary plasmids are separately transformed into A.tumefaciens cells. Following transformation, equal amounts of each typeof recombinant A. tumefaciens cells are co-infiltrated or introducedinto N. benthamiana leaves.

The pDUR22XLR1R expression system is tightly regulated and chemicallyinduced by estradiol. Upon application of estradiol, RNAs 1 and 2 ofpDUR22XLR1R expresses the replicase genes. The expression of RNA 1 andRNA 2 leads to the replication of the recombinant gene in the RNA 3deriving from the second binary plasmid comprising of the pQA series.

Example 3 Agroinfiltration and Induction of Transactivation

A. tumefaciens EHA105:pCH32 cells containing the appropriate plasmidswere grown for 24 to 48 hours in 2 ml LB broth. Approximately 0.5 ml wasthen transferred to 25 ml LB medium supplemented with 10 μl of 100 mMacetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) (AldrichChemicals, Milwaukee, Wis.) and 0.5 ml of MES buffer (pH 5.6) and grownovernight at 28° C. with shaking until cell density (OD₆₀₀) reached 1.0absorbance units. Cells were harvested by centrifuging at 2600 g,resuspended in 10 ml sterile de-ionized water, and cell density wasadjusted to 1.0 absorbance units. Five ml of the A. tumefaciens cellsuspension for each plasmid (p35S-SPAAT, pXVE-SPAAT, and pCMV-SPAAT) wasseparately mixed with either five ml of sterile water or A. tumefacienscells containing the p35S:p19 plasmid, then supplemented with magnesiumchloride to reach a final concentration of 10 mM and acetosyringone to150 μM and incubated at room temperature for three hours. Bacterialsuspensions were then pressure infiltrated onto the abaxial side ofyoung N. benthamiana leaves using a three ml sterile syringe without aneedle at 1-2 points to sufficiently cover at least half of the leaf(for information see the world wide web atwww.jic.bbsrc.ac.uk/sainsbury-lab/dcb/services/agroInfill.mpg).

About 12 hours after infiltration, a 50 μM solution of 17-β-estradiol(Sigma Inc., St. Louis, Mo.) in 0.05% Tween 20 was applied using cottontipped applicators to both sides of infiltrated N. benthamiana leaves.No solution was applied to plants to be used as non-induced (control) aswell as the p35S-SPAAT-infiltrated and healthy control plants.

Example 4 Protein Extraction, Quantification and Immunoblot Analysis ofExpressed Proteins

Plant leaves were sampled on the infiltrated area 2.5 days (60±1 hrs)post-infiltration by collecting four one cm diameter discs per leaf.Extraction buffer composed of 20 mM Tris-HCl (pH 8.1), 150 mM NaCl and0.01% (vol/vol) Tween 80 was added at 10 μl per mg fresh weight oftissue, and cells were lysed on ice using a plastic pestle. Lysate wascleared by centrifugation at 20,000 g for 20 min at 4° C. Thesupernatant was collected and stored on ice briefly until assayed. Totalsoluble protein concentrations were determined by the Bradford proteinassay (BioRad, Hercules, Calif.) using bovine serum albumin (BSA)(Fisher, Pittsburgh, Pa.) as the standard.

Example 5 Immunoblot Analysis

Proteins were denatured by boiling 40 μl supernatant samples for fiveminutes in 10 μl sodium dodecyl sulfate (SDS) sample buffer resulting ina final buffer concentration of 0.4% (vol/vol) SDS, 62.5 mM Tris-HCl (pH8.2), 10% glycerol, 5 mM EDTA and 0.01% (wt/vol) bromophenol blue. A 15μl aliquot of each denatured sample was fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% Tris-HClprecast gels (BioRad) carried out in a Mini-PROTEAN 3 electrophoresiscell (BioRad) for 35 min at 200 V. Proteins were then blotted onto 0.45μm nitrocellulose membranes (GE Osmonics, Minnetonka, Minn.) for 85 minat 120 V using an electroblot Mini Trans-Blot transfer cell (BioRad).Immunoblot analysis was then performed according to the proceduredescribed elsewhere (Huang et al., 2001) with the only alteration beingthe vendor of the secondary antibody used in this study (SouthernBiotechnology, Birmingham, Ala.). Chemiluminescence detection was usedfor the CMV CP immunoblot. Human AAT (Calbiochem, La Jolla, Calif.) wasused as the standard for all assays performed.

Example 6 Band Shift Assay

Formation of a covalent complex between AAT and porcine pancreaticelastase (PPE) (Wilczynska et al., 1997) was examined by visualinspection of functional AAT by immunoblot analysis. Briefly, 2 μl ofeither supernatant sample or human AAT standard (3.1 pmol AAT) was addedto 2 μl (100 pmol) of PPE (Calbiochem) in 36 μl extraction buffer andincubated for 20 min at 37° C. The complex was then analyzed using thesame immunoblot analysis previously described.

Example 7 Quantification of Total Recombinant AAT by ELISA

Rabbit anti-human α₁-antitrypsin polyclonal IgG fraction was diluted1:4,000 in phosphate buffer saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10.1mM Na₂HPO₄ and 1.8 mM KH₂PO₄, pH 7.4) was used to coat ELISA platesovernight (Corning, Corning, N.Y.). The wells were then blocked using200 μl of 0.1% (wt/vol) BSA in PBS and washed with PBS containing 0.05%(vol/vol) Tween 20 (PBST). Supernatant samples were diluted in PBST and50 μl was added per well and incubated for one hour at 25° C. Humanα₁-antitrypsin, diluted in PBST to concentrations from 800 ng/ml to 0.78ng/ml by two-fold dilutions, was used to generate the standard curve.After washing, 50 μl of horseradish peroxidase-conjugated polyclonalgoat anti-human α₁-antitrypsin IgG (HRP) (US Biological, Swampscott,Mass.) diluted 1:4,000 in PBS was added to the wells and incubated forone hour. The wells were washed and 100 μl of SureBlue peroxidasesubstrate solution (KPL, Gaithersburg, Md.) was incubated for 25 min.The reaction was stopped by the addition of 100 μl of HCl (1 M) and theabsorbance at 450 nm was measured with a SpectraMax 340 pc microplatereader (Molecular Devices, Sunnyvale, Calif.).

Example 8 Quantification of Functional Recombinant AAT

Using the principle of the band shift assay, 100 μl of PPE diluted1:1,500 in PBST was added in excess to 100 μl of either supernatantextract sample or human AAT standard (again diluted from 800 ng/ml to0.78 ng/ml) and incubated for 20 min at 37° C. to allow formation of theAAT-PPE complex. The same ELISA protocol was used as for total rAATdetection except that polyclonal rabbit anti-elastase IgG conjugated toHRP diluted 1:30,000 in PBS was used as the secondary antibody to allowspecific detection of the AAT-PPE complex. This allowed quantificationof the amount of functional recombinant AAT.

Example 9 CMViva Protein Expression in Plants

It was first determined if it is possible to obtain high levels ofprotein expression via agroinfiltration of non-transgenic plants withthe CMViva expression system using plasmid construct pCMV (FIG. 2), andif the protein expression could be controlled. The results showed thatCMViva gave very high expression of the native CMV CP after induction byadding estradiol (FIG. 3). By using the sensitive chemiluminescenceassay at saturation conditions it was possible to investigate if theexpression of the CMV CP was tightly controlled. The data showed thatCMViva-driven expression of CMV CP was slightly leaky as low levels ofCP were seen in plants in almost all experiments (FIG. 3, lane 7).However, upon induction with estradiol very high levels of CMV CP weredetected. In fact when compared on a wt:wt basis, similar levels of CMVCP were seen in CMViva-expressing N. benthamiana leaf tissues (FIG. 3,lanes 2-4) and CMV-infected Cucurbita pepo plants (FIG. 3, lane 1), anatural host for the CMV strain used herein.

The next experiment focused on whether one could use the CMViva systemto express desirable proteins in non-transgenic plants. The humantherapeutic protein AAT was chosen to further investigate this. Thequalitative and quantitative results were clear and dramatic. Acomparison focused on transient expression of rAAT in N. benthamianaleaves at 2.5 days post-infiltration using the CaMV 35S promoter(p35S-SPAAT), estradiol-driven Lex operator system (pXVE-SPAAT), and theCMViva system (pCMV-SPAAT). To achieve enhanced expression, the threesystems were evaluated when co-infiltrated with A. tumefacienscontaining the known gene silencing suppressor p19 (Voinnet et al.,2003). CMViva and pXVE-SPAAT were evaluated both with and withoutestradiol treatment to assess the ability to regulate proteinexpression.

Immunoblot analysis showed that all three systems gave detectable rAATaccumulation. rAAT expression via pXVE-SPAAT appeared to be slightlyleaky (FIG. 4A, lane 5) while higher levels of rAAT were detected uponinduction with estradiol (FIG. 4A, lanes 4 and 9). CMViva expression ofrAAT was greater than that for pXVE-SPAAT (FIG. 4A, lanes 6 and 10compared with lanes 4 and 9), and appeared to be more tightly regulatedsince very little rAAT was detected before addition of estradiol (lane7), while high levels of rAAT were seen after adding estradiol (lanes 6and 10). It should be noted that in this experiments the CMViva-producedrAAT seen in the immunoblots (FIG. 4A, lane 6) exhibited a slightlyfaster migration in SDS-PAGE than did human AAT used as a control (FIG.4A, lane 8). The rAAT produced in these studies had a lower molecularweight than authentic human AAT. This has been observed in otherplant-based expression systems, and has further been attributed to anumber of possible reasons including, lacking a portion of theC-terminal end of rAAT (Terashima et al., 1999), expression of differentforms of rAAT (Huang et al., 2001; Terashima et al., 1999), and/ordifferences in glycosylation patterns (Trexler et al., 2002). Therefore,in order to determine if the CMViva-produced rAAT exhibited functionalcharacteristics like the human AAT control, both of their abilities tobind to porcine pancreatic elastase (PPE) were compared. When AAT wasincubated with PPE, followed by SDS-PAGE and immunoblot analysis, AATbound with PPE exhibited a slower migration (band shift). FIG. 4Bpresents results of this analysis and shows that CMViva-expressed rAATresulted in a band shift as did the human AAT control (see lanes 7 and9), and it also suggested that the PPE-bound CMViva-expressed rAATprovides a high percentage of the total.

Example 10 Effect of the Gene Silencing Suppressor p19

The next assessment focused on whether co-infiltration with A.tumefaciens containing the gene silencing suppressor p19 enhanced therAAT production. It is known that agroinfiltration and CaMV-driventransient protein production in nontransgenic plants can be enhanced ifplant virus-encoded gene silencing suppressor proteins (SSPs) are usedto block post-transcriptional gene silencing (PTGS) in the infiltratedleaves. PTGS is a plant response resulting in degradation of specificRNAs, and many plant virus-encoded proteins have been shown to have theability to block PTGS. Therefore, the effect of p19 by co-infiltratingA. tumefaciens cells containing either p35S-SPAAT, pXVE-SPAAT orpCMV-SPAAT and A. tumefaciens cells containing either p35S:p19 orsterile water into N. benthamiana leaves were evaluated and therespective rAAT production was compared. Co-infiltration with p35S:p19resulted in higher rAAT levels for pXVE-SPAAT even when no estradiolinducer was added (FIG. 5A, lane 3 vs. lane 5) while more rAAT was seenfor treatments receiving both p19 and inducer (lane 2). MoreCMViva-produced rAAT was also achieved when using p19 (FIG. 5A, lane 6vs. lane 8). Here, CMViva production of rAAT was neither increased whenleaves were treated with p19 nor with estradiol inducer (FIG. 5A, lane7). The ability of CMViva-expressed rAAT to bind PPE was also evaluated.Treatments that included p19 and induction with estradiol showed themost rAAT bound with PPE (FIG. 5B). Thus, CMViva in combination with thep19 SSP resulted in the highest levels of functional rAAT (as assessedby binding with PPE).

Example 11 Quantification of Total and Functional rAAT

In order to take a more quantitative approach, ELISA was used todetermine total and functional rAAT in agroinfiltrated N. benthamianaleaf samples containing each of the three expression vectors. ThepXVE-SPAAT and pCMV-SPAAT systems were analyzed both with and withoutestradiol induction while all three systems were analyzed with andwithout p19. The total soluble protein (TSP) concentrations weremeasured and production of rAAT was expressed in terms of a percentageof the TSP concentration for each sample (FIG. 6).

Use of the constitutive CaMV 35S promoter alone resulted in transientproduction of total rAAT at 0.06±0.03% TSP and this level did notsignificantly change even with p19, which resulted in 0.05±0.01% TSP.The pXVE-SPAAT system showed detectable rAAT levels even withoutaddition of the estradiol inducer, resulting in total rAAT at 0.03±0.02%TSP without p19 and 0.12±0.05% TSP with p19. Induction of the pXVE-SPAATsystem increased total rAAT production, leading to 0.12±0.04% TSP and0.57±0.28% TSP without and with p19, respectively. The CMViva expressionsystem was tightly controlled when not induced, resulting in anundetectable level of total rAAT both without and with p19. The CMVivaexpression system with induction gave the highest levels of total rAAT,resulting in rAAT at 0.57±0.09% TSP without p19, a level similar topXVE-SPAAT with p19, and increased to 1.7±0.53% TSP with p19.

Quantification of functional rAAT was also measured by ELISA andrepresented in terms of a percentage of the TSP concentration. The CaMV35S promoter resulted in only small amounts of functional rAAT, at0.006±0.001% TSP without p19 and 0.007±0.001% TSP with p19. Just as forthe total rAAT, the pXVE-SPAAT system produced minimal functional rAATeven without addition of the estradiol inducer, resulting in functionalrAAT without and with p19 at 0.0013±0.0006% TSP and 0.0089±0.0010% TSP,respectively. Induction of the pXVE-SPAAT resulted in no detectablefunctional rAAT in the absence of p19 and 0.013±0.003% TSP in thepresence of p19. Induction of the CMViva expression system resulted inthe highest production of functional rAAT at 0.16±0.048% TSP without p19and 1.2±0.36% TSP with p19. Thus 70±3.3% of the total rAAT producedusing the CMViva expression system in combination with the p19 SSP wasin the functional form.

As can be seen in the examples above, the invention encompasses anefficiently regulated plant virus amplicon expression system (herereferred to as CMViva) for transient expression of proteins in plants.Furthermore, the usefulness of CMViva was demonstrated by qualitativeand quantitative analysis of a CMViva-produced human therapeuticprotein, AAT. The CMViva expression system described here isconceptually similar to the system described by Mori et al., (2001) butdiffers in several key aspects, resulting in important and distinctadvantages (FIG. 1A). Firstly, the use of the estradiol-inducible XVEsystem (Zuo et al., 2000) provides a distinct advantage. XVE is a3-component fusion protein comprised of a DNA binding moiety (bacterialrepressor LexA (X)), a transactivating domain VP16 (V) and theregulatory region of the human estrogen receptor (E), which functions astranscription activator. Using this system, Zuo et al., (2000) observedan eight-fold increase in GFP expression in transgenic Arabidopsisplants upon application of estradiol when compared to GFP expressionusing a constitutive CaMV 35S promoter system, and no detectable GFPtranscripts under non-induced conditions. The XVE system is more tightlyregulated with minimal “leakage” in the absence of inducer, estradiolhas no deleterious physiological effects on plants (as has been observedwith DEX), and estradiol is more soluble than DEX in aqueous solutions.In the experiments described herein, however, XVE-driven rAAT wasslightly leaky suggesting that the XVE system alone was not suitable forproviding controlled expression of desired proteins such as AAT.Secondly, we used CMV, rather than BMV (as used by Mori et al., (2001)).Like for BMV, the CMV genome is composed of three positive-sense,single-stranded RNAs. All three genomic components (RNA 1, RNA 2 and RNA3) are required for a competent infection, but unlike BMV, CMV encodesfor the potent silencing suppressor protein 2b. CMV RNA 2 encodes forthe 2b protein, but 2b is expressed from the 2b subgenomic RNA (sgRNA)which is derived from RNA 2 only after RNA replication, which occurshere upon addition of the estradiol inducer. Thirdly, the inventionencompasses modification of the 5′ sequence of the CMV1a cDNA so thatpositive sense 1a RNA will not be replicated in the presence offunctional replicase, as would normally occur in wild type CMV.Fourthly, all three CMV components were engineered on a single Tiplasmid in A. tumefaciens, thereby significantly decreasing the amountof time that would normally be required to generate and cross transgeniclines containing each component separately in order to obtain stablytransformed transgenic plants. Finally, the use of the CMViva expressionsystem as a transient production method using wild type N. benthamianaplants was demonstrated herein. This is particularly significant sinceit allows for rapid construct design, as well as rapid, efficientprotein production in non-transgenic plants.

It was found that while p19 had no effect on enhancing rAAT levels whenusing p35S-SPAAT, co-infiltration with p19 significantly improved totalrAAT production in both the induced XVE and CMViva systems. In theCMViva system, the impact of p19 was a three-fold increase in total rAATproduction, while in the XVE system a five-fold effect was observed. Theeffect of p19 was even more profound on the production of functionalrAAT resulting in an eight- and sixteen-fold increase for CMViva andXVE, respectively. The presence of p19 also increased the percentage offunctional rAAT relative to the total rAAT level, resulting in a maximumof 70±3.3% in this study. Notably, p19 addition enhanced rAAT expressioneven though the CMViva was designed to encode the CMV2b silencingsuppressor.

Thus, the experiments have shown that the CMViva system provides theability to tightly control the timing of gene expression and resultingprotein production even in non-transgenic plants. CMViva resulted inrAAT production which was 30 to 170-times greater than that obtainedusing the constitutive CaMV 35S promoter for total and functional targetprotein. The experiments have also shows that the CMViva amplicon systemprovides new opportunities for producing biologically active mammalianproteins in plants, and can even overcome some of the common problemsseen when using stably transformed plants.

The CMViva expression system may further be evaluated in transgenic cellcultures in order to develop a CMViva based production platform thatwill facilitate large-scale estradiol treatment for induction and easierrecovery of recombinant proteins secreted into the medium.

Example 12 CMViva is an Efficient Plant Expression System

As shown above, the novel Cucumber mosaic virus (CMV) inducible viralamplicon (CMViva) expression system allows for tightly regulatedchemically-inducible expression of heterologous genes in plant hosts.Transient production of recombinant α₁-antitrypsin (rAAT), a human bloodprotein, was shown in Nicotiana benthamiana leaves. The highestproduction levels were obtained by co-infiltrating leaves withAgrobacterium tumefaciens cells containing CMViva carrying the AAT geneand A. tumefaciens cells carrying a binary vector constitutivelyexpressing the gene silencing suppressor p19. Accumulation of up tothirty-fold more rAAT was observed in leaves (24 mg per 100 g leaftissue) when compared with the expression levels observed using the CaMV35S promoter. Significantly, 70% of the rAAT produced using the CMVivaexpression system was found to be biologically active, a 170-foldincrease in functional protein compared with the CaMV 35S expressionsystem.

Example 13 Production of Functional Recombinant Human Protein inTransgenic Tobacco Cell Cultures: Comparison of Gene Expression Systems

Three different types of expression systems, (1) the constitutive p35S,(2) the chemically inducible XVE, and (3) the chemically inducible viralamplicon CMViva, were compared for the production of human therapeuticprotein coded by the same transgene in transgenic N. benthamiana cellcultures in bioreactors. The following example provides the use of thechemically inducible XVE promoter system and the chemically inducibleviral amplicon CMViva expression system in stably transformed plant cellcultures for human protein expression.

Construction of the p35S-spAAT (35S), pXVE-spAAT (XVE) and pCMV-spAAT(CMViva) expression vectors is described in Example 1. The codon usageof the human AAT gene sequence was optimized according to the codonpreference of rice cell host (Oryza sativa). The three expressionsystems were stably transformed into Nicotiana benthamiana cells usingAgrobacterium-mediated transformation by the Agrobacterium tumefaciensstrain EHA105 pCH32 (Hamilton 1997) carrying appropriate binary vectors.Newly expanded leaves from N. benthamiana plants were cut into 1 cmsquare sections soaked in an Agrobacterium solution adjusted to 0.1 OD600 for 10 minutes and incubated on co-cultivation medium consisting ofMurashige and Skoog minimal organics (MSO) medium modified with 30 g/Lsucrose, 2 mg/L 6-benzylaminopurine (BA), and 200 μM acetosyringone, pH5.8, at 23° C. in the dark for 2˜3 days. Leaves were transferred toagar-solidified induction medium consisting of MSO medium modified with30 g/L sucrose, 2 mg/L BA, 400 mg/L carbenicillin, 250 mg/L cefotaxime,and 250 mg/L kanamycin and incubated at 26° C. for 10 days. Planttissues were subcultured until shoots formed. Shoots were harvested andtransferred to agar-solidified rooting medium consisting of halfstrength MSO medium modified with 15 g/L sucrose, 2 mg/L BA, 1.3 g/Lcalcium gluconate, 400 mg/L carbenicillin, 250 mg/L cefotaxime, and 100mg/L kanamycin. Leaves were removed from each rooted shoot with aportion of the petiole attached and placed on callus-generating mediumconsisting of MSO medium modified with 30 g/L sucrose, 0.4 mg/L2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin, 400 mg/Lcarbenicillin, 150 mg/L timentin, and 100 mg/L kanamycin for developingtransgenic callus. Transgenic callus was subcultured every 3-4 weeks andmaintained on agar-solidified KCMS medium consisting of 30 g/L sucrose,4.3 g/L MS salt mixture, 0.1 g/L myo-inositol, 0.204 g/L KH2PO4, 0.5mg/L nicotinic acid, 0.5 mg/L thiamine-HCl, 0.5 mg/L pyridoxine-HCl, 0.2mg/L 2,4-D, 0.1 mg/L kinetin, and 100 mg/L kanamycin, pH 5.8.Heterologous gene silencing suppressors were not incorporated into orapplied to any of systems.

Transgenic N. benthamiana cell lines were maintained in liquid KCMSmedium containing 100 mg/L kanamycin, pH 5.8. Transgenic N. benthamianacell suspensions were maintained in 250 mL flasks on an orbital shakerat 140 rpm and 25˜26° C. under ambient light and subcultured weekly bytransferring 10 mL into 90 mL fresh KCMS medium.

The inducer, 17-beta-estradiol, dissolved in DMSO, was added to culturesof XVE and CMViva transgenic cell lines at a specific time afterinoculation to start induction. The final concentration of DMSO was nothigher than 0.3%.

A BioFlo 3000 bioreactor (New Brunswick Scientific) with a singlepitched blade impeller containing 3.5 L KCMS medium was autoclaved.Inoculum cultures grown in 250 mL KCMS medium in 1 L Erlenmeyer flaskson an orbital shaker at 140 rpm, 25° C., for 7 days were combined toinoculate the bioreactor at a 10% density (volume of inoculatingsuspension to final volume). The bioreactor was maintained at 25° C.,50-75 rpm, and 40% (air saturation) dissolved oxygen under ambientlight. The dissolved oxygen concentration was controlled by manipulatingthe oxygen concentration in the gas sparging stream. Gas was introducedthrough a spherical gas diffusion stone with a pore size of 20 μmpositioned at the bottom of the bioreactor. The aeration rate wasregulated as necessary (0.4-1.0 L/min) for dissolved oxygen control.Oxygen uptake was monitored by measuring the change in dissolved oxygenin the absence of aeration. The pH was recorded on-line as well asoff-line for verification. At each sampling time, culture samples werecollected, placed in 1.5 mL microfuge tubes, and centrifuged at 14,000rpm for 10 min at 4° C. The supernatant was stored at −80° C. for lateranalysis.

Fresh cell weight (FCW) was measured by filtering 10 mL culture onto apredried, preweighed, Whatman #1 filter connected to a vacuum, washingthe cells with 20 mL ddH2O to remove residual sugars, then weighing thecells. Dry cell weight (DCW) was estimated after drying the retainedcells at 60° C. for 2 days.

An immunoassay based on competitive binding was developed forquantitative determination of estradiol in culture medium. Mouseanti-estradiol-6-carboxy methyloxine (6-CMO)-bovine serum albumin (BSA)monoclonal immunoglobulin G (IgG), the capture antibody, was diluted1:4,000 in phosphate-buffered saline (PBS), and ELISA plates were coatedwith it overnight at 4° C. The plates were washed with PBS containing0.05% (v/v) Tween 20 (PBST) before loading samples and standards.Estradiol standards at different concentrations (0 to 8000 pg/mL) andsamples diluted in PBST were placed in wells. Horseradishperoxidase-conjugated estradiol, diluted 1:500 in PBST, was dispensed toeach well and the plate was incubated at room temperature for 2 hours.SureBlue peroxidase substrate solution was added and incubated for 30min. The enzyme reaction was terminated by addition of 1 N HCl, andabsorbance at 450 nm was measured and recorded with a SpectraMax 340PCmicroplate reader.

Protease activity was quantified by a modification of a publishedprocedure (Joo et al. 2006). Substrate solution (0.3 mL) containing 10mg/mL casein sodium in 50 mM phosphate buffer (pH 7.0) was added to 0.3mL sample. The mixture of substrate and sample was incubated at 37° C.for 30 min, when the reaction was terminated by adding 0.3 mL 10%trichloroacetic acid. The mixture was allowed to stand for 10 min, thencentrifuged at 12,000×g for 5 min. Fifty μL supernatant, 100 μL 0.5 NNaOH, and 50 μL of diluted Folin & Ciocalteu's phenol reagent werepipetted into wells of a 96-well plate and incubated 30 min at roomtemperature. Absorbance at 660 nm was measured and recorded with aSpectraMax 340PC microplate reader. One unit of protease activity willhydrolyze casein to produce a color equivalent to one μg of tyrosine perminute at pH 7.0 at 37° C. (color by Folin & Ciocalteu's Reagent).

All transgenic callus lines were similar in color and consistency towild type N. benthamiana callus. Callus lines were subcultured monthlyand there were no changes in callus appearance over 1.5 years for theCMViva and XVE transgenic lines and over 1 year for the 35S line. Thehighly sensitive total and functional AAT ELISA and western blottingassays were used to screen and select transgenic cell lines producingrAAT. 33, 17 and 20 independently transformed callus lines with the 35S,XVE and CMViva expression systems, respectively, were screened for rAATproduction. Transgenic lines were placed in 6-well-plates containingKCMS medium for cell growth. After 5 days, estradiol was added at afinal concentration of 10 μM to induce AAT expression in the XVE andCMViva systems. The 35S callus lines were screened similarly, but noinducer was added. Preliminary ELISA screening for rAAT expressionidentified cell lines with high concentrations of extracellular rAAT.Extracellular rAAT yields varied widely among independent transformants(data not shown). Extracellular total rAAT was found above 100 μg/L in40% (14/33) of 35S lines, 12% (2/17) of XVE lines, and 10% (2/20) ofCMViva lines. Callus lines producing the most rAAT were established ascell suspensions in 250 mL Erlenmeyer flasks for secondary screening.After secondary screening, candidate cell lines expressing rAAT wereconfirmed by western blotting (data not shown). The cell lines withhighest yield of total and functional rAAT were CMViva-Line 8011,XVE-Line 6011 and 35S-Line 0632; these were selected for bioreactorstudies.

Western blotting and band shift analysis were used to confirm themolecular weight and functionality of expressed rAAT (FIG. 7). All threeexpression systems were successfully transformed into tobacco andproduced detectable extracellular rAAT in suspension cultures (FIG. 7).The rAAT produced by all three systems had a lower molecular weight (˜48kDa) than authentic human AAT (52 kDa) and for some samples, especiallyfrom XVE and 35S lines, multiple immunoreactive bands were observed.This could be due to lack of a C-terminal segment (Terashima et al.1999), different protein conformation (Huang et al. 2001), ordifferences in glycosylation (Trexler et al. 2002). No immunoreactivebands were detected in the absence of inducer but significantconcentrations of rAAT were produced by CMViva and XVE lines afterestradiol induction (FIG. 7). Therefore, both inducible systems weretightly regulated. Band shift analysis for functional rAAT showed acharacteristic lower MW immunoreactive band for the transgenic samplesfollowing PPE incubation, but the higher MW AAT-PPE complex was notdetected. Since band shift analysis detects concentrations of AATgreater than 200 μg/L, the more sensitive AAT ELISA was used insubsequent studies to determine the total and functional rAATconcentrations in the extracellular medium.

For inducible systems, the correct inducer concentration and timing ofapplication are critical to maximize recombinant protein expression. Twoinducible expression systems were evaluated for rAAT protein production.The concentration of inducer (COI) required for inducing maximum rAATproduction and the close-response of inducible plant cell cultures weredetermined. Inducer was added to the XVE and CMViva cell suspensions 10days after inoculation (100 mL cell culture in a 250 mL Erlenmeyerflask, 140 rpm and 25° C. under ambient light) to a final concentrationof 0, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50 or 100 μM. Samples were harvested8 days after induction and total and functional extracellular rAAT weredetermined by ELISA (FIG. 8). In the absence of inducer, rAAT was notdetected in XVE or CMViva cultures. There was a dose-dependent responsein rAAT expression in XVE and CMViva cell cultures with added inducer,and both inducible systems allowed regulation of rAAT expression atinducer concentrations between 0.1 and 10 μM. Above approximately 10 μMestradiol, rAAT production leveled off in both XVE and CMViva systems.The CMViva system first responded at 0.1 μM while XVE required 2.5 μM.Higher extracellular rAAT productivity was produced in CMViva at arelatively low inducer concentration (0.5 μM) compared with that in XVEsystem (5 μM) and CMViva produced more functional rAAT at all inducerconcentrations.

The timing of induction (TOI) relative to the culture growth phase wasinvestigated to maximize rAAT production. To induce rAAT proteinexpression at various cell physiological states, estradiol was added ata final concentration of 10 μM to 5 (early-exponential phase), 8(mid-exponential phase), 11 (late-exponential phase) or 14 (stationaryphase) day old cultures of XVE and CMViva. Induction of rAAT productionin XVE cells was similar over the growth cycle, although culturesinduced at day 5 had a 2 day lag phase (FIG. 9). Higher extracellulartotal rAAT was seen with later induction, but this system produced verylittle extracellular function rAAT.

The CMViva system had a similar lag phase for extracellular total rAATproduction when induced at day 5 and timing also affected extracellularfunctional rAAT production (FIG. 10). There was more extracellularfunctional rAAT in cultures induced at later growth stages, with thegreatest yield at 11 days after inoculation. Although total rAAT wassecreted and detected by ELISA shortly after applying the inducer,extracellular functional rAAT was not detectable until day 16-18 for allcultures. This implies that some factor in CMViva cell culture mayimprove functional rAAT protein production or stabilize itsfunctionality in plant cell culture. The yield of extracellularfunctional rAAT was greater in CMViva cultures than in XVE cultures,although total rAAT was lower.

Based on the above results, the bioreactor studies induced proteinsynthesis with 10 μM estradiol at 11 days after inoculation. Cell growth(biomass concentration in DCW and FCW), pH, oxygen uptake rate (OUR),extracellular estradiol concentration, total and functionalextracellular rAAT concentration and yield of functional rAAT intransgenic plant cell cultures were examined using each of the threegene expression systems and in wild type N. benthamiana.

The cell growth and oxygen uptake rate (OUR) for XVE and CMViva cultureswere similar to that of wild type N. benthamiana cell culture (FIG. 11,Table 2). The 35S system had a longer lag phase and a lower OUR. Thespecific growth rate, doubling time, OUR and biomass accumulation in thegrowth phase of XVE and CMViva cultures were significantly superior to35S cultures. This implies that cell growth of 35S cultures wasinhibited by constitutive rAAT production during the cell growth phase.Interestingly, all three transgenic cultures, but not the wild typecontrol former larger cell aggregates in late stationary phase (data notshown). Extracellular protease activity also varied among geneexpression systems. CMViva and wild type cultures produced the most andleast extracellular protease activity respectively (Table 2), CMVivacultures also exhibited the highest FCW (450 g-FCW/L) and lowest ratioof DCW to FCW (˜3%) (FIG. 11). Extracellular total soluble protein forthe three systems was between 143 and 197 mg/L (data not shown). Theextracellular pH of 35S and XVE cultures dropped from the initial pH ofapproximately 5.8 and then gradually increased at day 1 afterinoculation. However, the pH increase during induction of CMVivacultures was faster than for the 35S or XVE (FIG. 11).

The yield and functionality of rAAT produced by the three transgeniccultures were measured by total and functional AAT ELISA. The CMVivatransgenic line had the highest titers of functional rAAT: 22% of thetotal rAAT expressed was found to be functional, defined as capable ofirreversible binding to porcine pancreatic elastase, PPE (FIG. 12).Furthermore, rAAT production reached a maximum in the CMViva systemfaster than in the other systems. The rAAT titers did not decline, evenduring the late stationary phase, in any of the three systems, but totaland functional rAAT leveled off for CMViva around 5 days afterinduction. The functional extracellular rAAT in 35S and XVE cultures wasvery low, close to the detection limit. In the absence of inducer, rAATwas not detected in XVE and CMViva cultures, demonstrating tightregulation. Estradiol was quickly depleted from the culture medium afterinduction (from 9.3 μM to 0.8 μM) but 2 days after induction theestradiol concentration in the culture medium gradually increased (FIG.12). It is not clear what caused either the rapid uptake or the slowrelease of estradiol to the culture medium. To elucidate the possiblemechanism for this concentration change, 10 estradiol was added to aflask of wild type N. benthamiana cells 10 days after inoculation, andthe kinetics of cell growth and extracellular estradiol concentrationwere determined (FIG. 13). Interestingly, the extracellular estradiolconcentration remained relatively stable at 7.84-9.08 μM. This suggeststhat the estradiol depletion and slow release in XVE and CMViva culturesis not due to physical factors such as surface adsorption to cellaggregates and/or estradiol degradation, but may represent intracellularuptake of estradiol by the XVE and CMViva cells.

Detectable rAAT expression was seen in the XVE and CMViva systems atestradiol concentrations as low as 100 nM (the lowest concentrationtested) and it reached saturation at ˜10 μM estradiol. Previous studiesusing the XVE system found detectable GFP transcripts in plants inducedwith 8 nM estradiol (Zuo et al. 2000) and GFP fluorescence in viralamplicon plant cell cultures induced with 10 nM estradiol (Dohi et al.).Saturation was reached around 5 μM estradiol for GFP transcripts inwhole plants and 0.1-1 μM estradiol for GPF fluorescence using the viralamplicon system in plant cell culture. Since rAAT titers level off atestradiol concentrations above 10 μM, some other factor may becomelimiting such as the constitutively produced XVE fusion protein. Theconcentration-dependent induction of XVE and CMViva cultures, suggeststhat rAAT expression can be regulated by estradiol concentrationsbetween 0.1 and 10 μM. Although this was expected for XVE, a binaryresponse (off without estradiol and fully on with estradiol) would beexpected for a truly autocatalytic viral amplicon system. However, inthe CMViva system, CMV1a transcripts are not replicated in the presenceof viral replicase due to the 57-nucleotide deletion at the 5′ end ofthe CMV RNA1 cDNA. Thus the CMViva system is not autocatalytic sinceCMV1a transcription is controlled by the XVE promoter, independent offunctional replicase. This makes the CMViva system less prone toleakiness and its expression is more controllable. The CMViva system wasmore sensitive to estradiol than the XVE system, producing detectablerAAT at just 0.1 μM estradiol instead of 2.5 μM. The XVE promoter wastightly regulated with no rAAT detected in the absence of estradiol andestradiol caused no obvious detrimental, toxic, or growth-inhibitingeffects on N. benthamiana cultures at up to 100 μM. Although 10 μMestradiol was adequate for batch suspension cultures in flasks, higherestradiol concentrations and multiple or continuous applications maybenefit fed-batch cultures or other high cell density operational modes.

Timing of induction is also important to optimizing productivity ofchemically inducible expression systems. Since heterologous geneexpression can significantly impact cell physiology and growth,induction during the early stage of culture may limit production due tolower biomass. Post-transcriptional gene silencing responses may also bestronger during early and mid-exponential growth states. During the latestationary phase cells may already be nutrient deprived, under stress,slowing metabolism/biosynthesis, secreting proteases and/or dying.Maximum total and functional rAAT were obtained when the inducer wasadded during the late exponential phase of growth, 11 days afterinoculation.

The performance of transgenic N. benthamiana cultures with three geneexpression systems were evaluated in bioreactors for cell growth andhuman rAAT production yield and functionality. The maximum specificgrowth rates, oxygen uptake rates and biomass concentrations obtainedwith XVE and CMViva cultures were similar to the wild type cultures(Table 2, FIG. 11), indicating that the chemically inducible cultureswere physiologically indistinguishable from the wild type cultures priorto induction. The fact that the XVE and CMViva callus and cellsuspension were subcultured for over 18 months with no observablechanges in phenotype or differences from wild type callus/cell linesalso supports this conclusion. No recombinant AAT was detected in theXVE or CMViva culture broths prior to induction, indicating tightregulation and negligible leakiness for the XVE promoter. Viral RNA andproduct (GFP) were not detected in an inducible tomato mosaic viralamplicon system using the XVE promoter in stably transformed tobaccoBY-2 cells (Dohi et al. 2006). The 35S culture had a lag phase and themaximum specific growth rate and oxygen uptake rate were significantlylower than for the wild type and other transgenic lines, presumably aresult of the metabolic burden associated with constitutive rAATexpression.

Following induction, the CMViva culture had more extracellular proteaseactivity and a more rapid pH increase than the other transgenic celllines. Heterologous protein expression by the CMViva viral amplicon mayrequire more energy to produce target protein and/or affect the plantcell physiology. In an ethanol-inducible plant DNA viral ampliconexpression system, excessive accumulation of either Rep (the viralreplication initiator protein) or viral amplicon components during thelate stage of induction had a toxic effect on tobacco NT1 cells (Zhangand Mason 2006).

As shown in FIG. 12, there were significant differences in the kineticsof rAAT production and the total and functional extracellular rAATobserved for the three transgenic cultures. Although the 35S and XVEcell lines had higher levels of extracellular total rAAT, the levels offunctional rAAT were very low or undetectable in these cultures. Thehighest extracellular concentration of functional rAAT (27 μg/L) andhighest ratio of functional rAAT to total rAAT (22%) were observed fromthe CMViva plant cell cultures four days after induction. It appearsthat functional rAAT is relatively unstable in the tobacco culturemedium and the accumulation of functional rAAT may depend strongly onthe production kinetics relative to the kinetics of reactions involvedin the loss of bioactivity. The production of extracellular rAAT in theCMViva culture indicates that functional viral replicase is producedafter induction and that the secretion signal peptide is effective intargeting the product to the culture medium. The relatively high levelsof functional rAAT suggests that the intracellular replication machineryinherent in the CMViva expression system may be beneficial foramplifying the mRNA of the target gene (AAT). In addition, higherfunctional rAAT yield might be also contributed to by the 2b protein, apotent silencing suppressor in CMViva system, which may decrease PTGS.However, the CMViva system produced the lowest level of extracellulartotal rAAT. It should be noted that the CMViva cell cultures in abioreactor exhibited the highest FCW and the lowest ratio of DCW to FCWduring the induction phase (FIG. 11). Plant cells from a phase of rapidcell division usually have smaller size, smaller vacuoles and lowerwater content than cells from the stationary growth phase. This suggeststhat the vacuoles in CMViva cell cultures may contribute to the FCW ofplant cells. Plant vacuoles contain a variety of proteases that areactive under mildly acidic conditions that may be released upon celldeath. Furthermore, the extracellular protease activity level duringCMViva cell cultures is higher than in XVE and 35S cell cultures (Table2). Thus, a more rapid cell death following induction coupled withhigher protease levels might explain why CMViva cell cultures producedlower total rAAT yield than the XVE and 35S cell cultures. Although weselected the highest producing cell lines for each expression system,differences in positional effects of T-DNA integration between thedifferent transgenic lines may also impact expression levels (Wilson etal. 1990).

In this example, an immunoassay was utilized for the quantitativekinetic analysis of estradiol in the chemically inducible plant cellcultures (XVE and CMViva system). Estradiol binds to the XVE fusionprotein which causes the XVE protein to dimerize and function as atranscriptional activator for the LexA promoter. The data show thatestradiol is rapidly depleted from the XVE and CMViva culture brothswithin 24 hours after it is introduced (FIG. 12). Interestingly,estradiol is not depleted from the culture broth when added to wild typeN. benth cultures, suggesting that the depletion from the culture mediumis not due to physical processes (e.g. adsorption to cell aggregates,instability of estradiol in the culture broth, etc) but may be due tointracellular uptake of estradiol by the XVE and CMViva cells. Estradiolis then gradually released into extracellular medium over the next tendays (FIG. 12). This release may be due to cell lysis and/or diffusionof estradiol from the cell once it is no longer bound to XVE.

In summary, the novel inducible plant viral amplicon system (CMViva),previously demonstrated by Agrobacterium-mediated transient expressionin N. benthamiana leaf tissue (see previous examples), can also be usedfor production of functional rAAT in stably transformed to N.benthamiana cells in bioreactors. Similarly to the results obtainedusing transient expression in N. benthamiana leaf tissue, we obtainedthe highest levels of extracellular functional rAAT with the CMVivaexpression system when compared to a constitutive CaMV 35S system andthe chemically XVE system. Table 3 compares recombinant AAT productionusing CMViva system by transient expression in N. benthamiana leaveswith and without co-infiltration with the p19 gene silencing suppressor,and transgenic N. benthamiana cell culture in a bioreactor in this study(without p19 expression). Functional rAAT production was 8-fold lower ona % TSP basis and 100-fold lower on a % DCW basis for the transgenicCMViva cell culture compared to transient agroinfiltration in plantleaves without the p19 gene silencing suppressor. However, the ratio offunctional rAAT to total rAAT was similar, 22˜28%, for both systems.There are several possible explanations for the differences inproductivity observed between the transgenic cell culture and transientleaf-based production systems. First, only extracellular rAAT wasmeasured in the transgenic cell culture analysis; cellaggregate-associated and/or intracellular rAAT was not included. For thetransient system, rAAT was measured in extracts of leaf tissue.Secondly, there may be significant differences in the stability of rAATin the leaf apoplast versus the plant cell culture medium due todifferences in concentration, type and activity of proteolytic enzymesas well as physiochemical differences (e.g. pH, composition, etc).Thirdly, inherent differences in the mechanisms involved in transientexpression versus expression from a stably integrated transgene may beresponsible. Other investigators have reported higher productivity usingtransient expression compared with stably transformed lines (Wroblewskiet al. 2005), attributing the differences to the relative timing of theonset of PTGS compared to transgene expression. Since the stablytransformed CMViva cultures constitutively express a number ofheterologous proteins (XVE, CMV2a, and CMV3a) it may be that PTGS hasalready been established prior to induction. Although the CMV2b proteinis known to be a potent gene silencing suppressor, since it is coded bya subgenomic RNA, it will only be produced after induction. Furthermore,previous examples demonstrated that co-infiltration with agrobacteriumcontaining an expression cassette for p19 could significantly improvethe rAAT production yield and quality (Table 3). Therefore, it may bepossible to enhance productivity in the transgenic CMViva cell linethrough constitutive expression of p19, CMV2b or other gene silencingsuppressors or by transient agroinfiltration of the CMViva cellaggregates for expression of gene silencing suppressors prior toinduction.

Example 14 Production of Functional Recombinant Human Protein inTransgenic Tobacco Cell Cultures: Culture Strategy of ImprovingHeterologous Protein Production

Changes in culture pH were applied during recombinant protein productionas a strategy to minimize the production, secretion and/or activity ofextracellular proteases capable of degrading extracellular recombinantprotein products. The following example demonstrates an inducibleexpression of a replicable viral amplicon for human protein productionin transgenic tobacco cell cultures in bioreactor and for the beneficialinfluences of pH on the production yield and functionality ofrecombinant human protein.

The p35S-spAAT (35S), pXVE-spAAT (XVE) and pCMV-spAAT (CMViva)expression vector constructions are described in previous examples.Three different expression systems have been stably transformed into N.benthamiana cells using Agrobacterium-mediated transformation by theAgrobacterium tumefaciens strain EHA105 carrying the appropriate binaryvectors as described in example 11.

Transgenic N. benthamiana suspension cell cultures transformed with oneof three gene expression systems expressing human AAT protein wereestablished and screened as described in the previous examples. Celllines with the highest extracellular titers, 35S line #0632, XVE line#6011 and CMViva line #8011, were selected for further study. TransgenicN. benthamiana cell cultures were maintained in KCMS medium, whichconsists of 30 g/L sucrose, 4.3 g/L MS salt mixture, 0.1 g/Lmyo-inositol, 0.204 g/L KH2PO4, 0.5 mg/L nicotinic acid, 0.5 mg/Lthiamine-HCl, 0.5 mg/L pyridoxine-HCl, 0.2 mg/L 2,4-D, and 0.1 mg/Lkinetin and was adjusted to pH 5.8 with KOH prior to autoclaving at 121□for 26 min, containing 100 mg/L kanamycin and subcultured weekly bytransferring 10 mL of established suspension cells into 90 mL KCMSmedium in a 250 mL flask at 140 rpm and 25□ under ambient light.

17-beta-estradiol prepared in DMSO as a chemical inducer solution wasadded to plant cell cultures (for the XVE and CMViva transgenic celllines) at a specific time after inoculation at a final concentration of10 μM to start the induction phase. No inducer solution was applied towild type (untransformed) N. benthamiana cell culture as negativecontrol or to transgenic plant cell cultures with the 35S constitutivepromoter.

Plant cell cultures were grown in a New Brunswick Scientific BioFlo 3000bioreactor (3.6 L working volume) with basic environmental conditionsset as described in example 11.

Before the induction phase, medium exchanges were performed byconnecting sterile tubing from an autoclaved bottle to the bioreactor. Aperistaltic pump was used to draw cultured medium from the bioreactorinto a bottle at a speed of approximately 50˜60 mL/min (agitation andaeration were kept on during the medium removal). A ring sparger with 11holes (˜1 mm diameter) was positioned close to the bottom of thebioreactor to filter out the medium while retaining the plant cells inthe bioreactor. After removing most of cultured medium (about 1,800 mL),an equivalent volume of fresh sterile KCMS medium was pumped into thebioreactor. After the inducer solution prepared in DMSO was added intobioreactor at a final concentration of 10 μM for the inducible promotersystems (XVE and CMViva), 0.5 N NaOH was added periodically to keep theculture pH higher than 6.4 (one-sided pH control) using base-additionmanual mode through the operator interface of the New BrunswickScientific BioFlo 3000 bioreactor. No medium exchange and inductionstrategies were applied to plant cell cultures with the constitute 35Spromoter but pH control was applied at specific time after inoculation.

Fresh cell weight (FCW) was measured by filtering a 10 mL sample onto apre-dried, pre-weighed, Whatman #1 filter connected to a vacuum andwashing the cells with 20 mL of ddH2O to remove residual sugars from thecell surfaces, then weighing the cells. Dry cell weight (DCW) wasestimated after drying the retained cells at 60 deg. C. for 2 days.

The methods of protein analysis including total soluble protein (TSP),western blotting, band shift assay for functional AAT analysis, totalAAT ELISA and functional AAT ELISA were performed as previouslydescribed in previous examples.

The concentration of estradiol in cell culture medium was measured by animmunoassay based on the principle of competitive binding as describedin the previous examples.

The protease activity in cell culture medium was quantified by accordingto a procedure which has been described in the previous examples.

For the investigation of human AAT stability in plant cell cultures,human AAT was added to the cell-free conditioned medium to a finalconcentration of 2 mg/L and distributed into 6-well-plates. Thecell-free conditioned medium was prepared by centrifuging and filtering(through a 0.22 μm filter) the plant cells from 7 day old CMViva cellcultures (pH 5.1). Various protease inhibitors including2-mercaptoethylamine-HCl (aminopeptidase inhibitor), AEBSF (serine andcysteine proteases inhibitor), benzamidine-HCl (serine proteaseinhibitor), and EDTA (a metal ion chelator and metalloproteinaseinhibitor) were added to the conditioned medium to a final concentrationof 10 mM to assess their ability to retard the degradation of human AAT.In another treatment, the conditioned medium was also heated (in boilingwater for 30 minutes) or the medium pH was adjusted (from original pH5.1 to 6.4˜6.6) prior to the addition of human AAT to investigate thehuman AAT stability. The 6-well-plates containing human AAT in variousconditioned medium (4 mL/well) were incubated on an orbital shaker at140 rpm and 25□ under ambient light. Samples were taken daily and storedat −80° for further ELISA, western blotting and band shift analysis.

To understand the recombinant AAT protein stability in plant cellcultures, commercially available human AAT was spiked into variouscell-free conditioned culture media to determine the total andfunctional human AAT concentration change over time. A preliminary studywas performed to examine the stability of human AAT in cell-freecultured medium prepared from 7 day old wild type N. benthamiana ortransgenic CMViva cell cultures. Western blot and AAT ELISA analyseswere applied to examine the degradation of human AAT over time (FIG.14). The human AAT was unstable in cultured medium (free of cells) anddegraded very quickly in both of the tobacco cell culture media. ELISAassays showed that after 1 day, there was only approximately 23% of theinitial functional human AAT remaining in wild type cell culture mediumand no detectable human AAT in CMViva cell culture medium. Theimmunoblot analysis showed a concomitant reduction in the intensity ofthe highest immunoreactive band and a corresponding increase inintensity of a lower immunoreactive band for the wild type N. benthculture broth, which corresponded well with the loss of functional humanAAT protein. For the CMViva culture broth two immunoreactive bands wereobserved even after 0.5 days and the intensity of both bands dropped offdramatically from 0.5 to 1 day incubation. It should be noted that somedegradation products were observed at lower molecular weight.

Various protease inhibitors and other strategies were investigated toenhance the stability of human AAT in cultured medium. The stability ofhuman AAT in cell-free plant cell culture medium with various treatmentsis shown in FIG. 15. It is interesting to note that functional human AATlevels decline by almost 50% over a 5 day incubation even in fresh,sterile KCMS medium at a pH of 5.8, while the total human AAT leveldeclines slightly (about 25%) over that time period. In the 7 day oldcell-free CMViva culture medium (which typically has a pH about 5.0),the functional human AAT is negligible within 2 days, while the totalhuman AAT level declines by 60% over the 5 day incubation period. Whenthe CMViva culture medium was adjusted to pH 6.4 the stability of humanAAT protein improved, with 40% of the functional AAT remaining at day 2and about 20% remaining at day 5. It should also be noticed that thetotal human AAT only declines about 25% over the 5 day period at thehigher pH, compared with the 60% loss at pH ˜5.0. These data suggestthat culture medium pH influences human AAT stability; increased pHslows the rate of degradation of functional AAT in the spent CMViva cellculture medium. In addition, the functional human AAT was much morestable in the heated CMViva culture medium with functional human AATmaintained as high as 70% of the initial concentration after 5 days.This suggests that some heat-labile media components, perhapsproteolytic enzymes, may be present in the cultured medium. Differenttypes of protease inhibitors (serine and cysteine proteases,aminopeptidase, and metalloproteinase inhibitors) were added to culturedmedium to a final concentration of 10 mM to assess whether proteaseinhibitors alone could retard human AAT degradation. The addition of thecysteine and serine protease inhibitor (AEBSF) showed the highestability to stabilize functional human AAT levels with results similar toincreasing the medium pH at 6.4. It therefore indicated that proteasesin the cell cultured medium may be contributing to the observeddegradation of human AAT and also implied that at least serine andcysteine proteases are involved in the human AAT degradation. Mediumadditives other than protease inhibitors including bacitracin, PVP,mannitol and BSA, have also been examined. However, these stabilizingagents did not show significant effect on stabilizing the functionalhuman AAT protein (data not shown). Although other papers have shownthat medium additives or stabilizing agents can improve recombinantprotein production, these data imply that pH is an important factor foraffecting the stability of human proteins. FIG. 2 clearly shows that thetotal human AAT in cultured medium at pH 6.4 was relatively stable andthe functionality of human AAT was degraded slowly compared with that inconditioned medium with an original pH 5.1 or in media containing otherprotease inhibitors. These results indicated that culture medium withhigher pH may decrease protease activity and/or help maintain thestructure and functionality of human AAT.

Western blots and band shift assays were applied to examine thestability and functionality of human AAT in various conditioned medium(FIGS. 16A and 16B). As shown in FIG. 16A, it was obvious that human AATin heated conditioned medium (FIG. 16A, lane 4) and conditioned mediumat pH 6.4 (FIG. 16A, lane 3) showed a higher AAT level (more intensehighest band) than both that in original conditioned medium at pH 5.1(FIG. 16A, lane 9) and in conditioned medium containing various proteaseinhibitors and/or protein stabilizers (FIG. 16A, lane 5˜8). Of thevarious protease inhibitors, the sample containing AEBSF had the mostintense top band. To examine the functionality of human AAT, band shiftanalysis was applied. FIG. 16B shows that human AAT in the heatedconditioned medium (FIG. 16B, lane 4) and conditioned medium at pH 6.4(FIG. 16B, lane 3) exhibited a significant band shift at 75 kDa (AAT-PPEcovalent complex, a reaction product of AAT binding to PPE) and a lowerband (the cleaved product of the AAT-PPE reaction), as did the human AATcontrol (FIG. 16B, lane 1), but the characteristic band shift was notshown in original conditioned medium (FIG. 16B, lane 9) and was lighterin conditioned medium containing various protease inhibitors (FIG. 16B,lane 5˜8). Of the various protease inhibitors, the sample containingAEBSF had the strongest immunoreactive band corresponding to the AAT-PPEcomplex. These data showed that the proteolytic degradation of human AATin cultured medium was evident and pH is an important factor foraffecting the stability and functionality of human AAT in cell culturemedium.

To further understand the pH protective effect on making human AAT lesssusceptible to proteolytic degradation or stabilizing human AAT in plantcell culture medium, the stability of human AAT was investigated inconditioned medium collected from different CMViva cell cultures atvarious ages. Human AAT was added to cell-free cultured medium at 7, 11and 16 days old post-inoculation, collected during the exponentialphase, early stationary phase and late stationary phase, respectively,of CMViva cell cultures grown in flask to evaluate the effect of culturebroth components on human AAT stability. Interestingly, as shown in FIG.17, the kinetics of human AAT degradation varied significantly indifferent age cell-free cultured medium. Functional human AAT wasdegraded quickly in 7 day-old cultured medium (pH 5.1) but wasrelatively stable in 11 day-old conditioned medium (pH 5.9) and muchmore stable in 16 day-old conditioned medium (pH 6.9). This isparticularly noteworthy as we would expect that the levels ofextracellular proteases would increase with culture age. These datasuggest that higher medium pH is favorable to stabilize functional humanAAT and/or reduces the activity of medium factors contributing to humanAAT degradation. To understand the effect of medium pH on human AATstability, human AAT was added to 11 day-old cell-free CMViva culturedmedium, which was adjusted to different pH values (4, 5, 6, 7 or 8). Thedegradation kinetics of human AAT in cultured medium with various mediumpH is shown in FIG. 18. It was found that higher medium pH (7˜8)maintains the functionality of human AAT but lower medium pH (4˜5)accelerates the loss of functionality. Accordingly, increasing themedium pH could increase the stability and functionality of human AAT inthe cell culture medium, presumably by decreasing the activity ofproteolytic factors, which are responsible to human AAT degradation incultured medium, and/or providing a favorable environment for thecorrect conformation of human AAT, which could execute its functionalityfor binding elastase.

Since medium pH plays an important role in stabilizing extracellularhuman AAT, we investigated whether increasing the culture pH during theprotein production phase in the transgenic plant cell cultures couldimprove the functional rAAT production yield. An induction medium with ahigher pH may decrease protease activity and/or maintain thefunctionality of recombinant AAT. According to this hypothesis, arational induction strategy was proposed to improve the total andfunctional rAAT production by performing a medium exchange beforeinduction (to remove most of the proteases in the cell culture medium)and also increasing the medium pH (to decrease protease activity and/orstabilize the conformation of functional AAT) during the proteinproduction phase.

An initial study was first performed to assess the effect of mediumexchange and medium pH on rAAT production in the CMViva system in shakeflasks comparing 1) adding the inducer only, 2) performing a mediumexchange before induction and then adding the inducer, and 3) performinga medium exchange before induction, then adding the inducer andadjusting the pH by adding NaOH to maintain the pH between 6.4˜6.8 every12 hours, respectively (FIG. 19). During the induction phase for thecase of the medium exchange without pH control, the pH profile wasfairly stable and relatively low (5.1˜5.6). Although medium exchange wasexpected to remove most of extracellular proteases, the total andfunctional rAAT was at the lowest level compared with other inductionstrategies. When the pH is adjusted to a higher level during theinduction phase following a medium exchange, the total and functionalrAAT yield was increased and the ratio of functional rAAT to total rAATwas enhanced from 0% (medium exchange, induction and without pH control)to 40% (medium exchange, induction and pH control). Thus, a culturemedium with higher pH during the protein production phase can improvefunctional rAAT productivity. Consequently, pH is an important variable,which exhibits a strong influence on the stability of human proteins forprocess optimization of plant cell cultures.

To confirm that the pH control strategy can be realized during theprotein production phase in plant cell cultures in bioreactors, theeffect of implementing a pH shift on rAAT production was investigated inall three systems (35S, XVE and CMViva). Medium exchange was performedbefore induction and pH adjustment in the bioreactor for the XVE andCMViva systems to provide additional nutrients. FIGS. 20, 21, and 22show the effect of pH control on total and functional rAAT productionfor the XVE, CMViva and 35S systems, respectively. Significantly, ineach system, functional rAAT titers improved dramatically when the pHduring the production phase was maintained at a higher value. In allcases, the total rAAT titers increased as well as the functional rAATtiter. With pH control, the CMViva cell culture achieved a higher levelof extracellular functional rAAT (100 ng/mL) and a higher ratio offunctional rAAT to total rAAT (48%) in four days after induction thanfor the XVE system (60 ng/mL and 7.1%). For the 35S system, two pHprofiles were applied to the cell culture at day 12 after inoculation(FIG. 22). These data showed that functional and total rAAT productioncould be further improved at higher pH (6.8) and that the pH shift didnot significantly inhibit plant cells growth. Thus, pH control duringprotein production is useful strategy for improving the production yieldand functionality of recombinant human protein in plant cell culture.

The results show that human AAT is unstable and is degraded rapidly inmedia in which wild type and transgenic N. benthamiana plant cells havebeen grown. To elucidate the mechanism of the loss of functional AAT inplant cell cultures, experiments with human AAT spiked into fresh KCMSmedium, heated cultured medium and other cell-free conditioned mediumcontaining protease inhibitors or altered pH demonstrated that the heatlabile components, most likely plant cell derived proteases, in themedium were primarily responsible for the loss or degradation of humanAAT in cultured medium. The results further suggest that higher mediumpH may decrease protease activity and/or stabilize human AAT proteinconformation facilitating it's binding to elastase (PPE) or reducingit's susceptibility for binding and/or cleavage by proteases.Additionally, the serine and cysteine protease inhibitor (AEBSF) andaminopeptidase inhibitor (2-mercaptoethylamine-HCl) could slightlyinhibit human AAT degradation in the cell cultured medium (FIGS. 15, 16Aand 16B), indicating that serine and cysteine proteases oraminopeptidase present in the culture medium may contribute to the humanAAT degradation. Work by Schiermeyer and coworkers (Schiermeyer et al.2005) indicates that metalloproteases are responsible for the DSPAα1protein degradation in tobacco cell culture medium and can be inhibitedby the addition of EDTA. The effect of stabilizing agents (bacitracin,PVP, BSA, Pluronic F-68, and mannitol) and protease inhibitors(2-mercaptoethylamine-HCl, AEBSF, benzamidine-HCl and EDTA) on the rAATproduction in transgenic plant cell cultures was also evaluated.However, no significant improvement on total and functional rAATproduction was achieved (data not shown). Thus, the ability ofstabilizing agents or protease inhibitors to inhibit target proteindegradation has to be evaluated on a case-by-case basis.

When the culture pH in the bioreactor is maintained at a higher levelduring production, the levels of functional extracellular rAAT aresignificantly enhanced in all of the transgenic plant cell culturesstudied (FIGS. 20, 21, and 22). This further suggests that functionalrAAT was successfully translated, folded and secreted into extracellularmedium and rAAT produced in transgenic tobacco cell cultures wasdegraded due to the proteolysis in the cell culture medium irrespectiveof the gene expression system used to express the rAAT protein. Theseresults demonstrate an effective strategy for improving human proteinproduction yield and quality by using optimized culture conditions andaltered environmental conditions during the protein production phase,such as increasing the culture pH, as an alternative to adding proteaseinhibitors or protein stabilizing agents in plant cell culture.

A recent study (Becerra-Arteaga et al. 2006) indicated the possibilitythat thioredoxin, a disulfide reducing protein, could be a destabilizingfactor that contributes to the denaturation of extracellular recombinantproteins in tobacco cell cultures, however AAT has no disulfide bonds.AAT is a member of the serpins superfamiliy (serine protease inhibitors)in which membership is based on the presence of a single common coredomain consisting of 3 β-sheets, 8-9 α-helices and a reactive centerloop (RCL) (Gettings et al., 2002). AAT inhibits its target protease(such as tripsin or elastase) by forming a stable and irreversiblecovalent binding complex in which the Met358-Ser359 bond in the RCL ofAAT is cleaved and the RCL is inserted into a β-sheet of the AAT(Huntington et al., 2000). The structure of RCL is the most variableregion and is crucial for the activity of AAT. In addition, human AAT isa metastable and conformationally flexible protein and contains ninemethionines and a cysteine in its primary sequence. Oxidative damage ofmethionines or cysteine in the protein active site to a sulfoxidederivative has been reported to result in a loss of inhibitory activityagainst elastase of AAT (Griffiths et al., 2002). The methionines in AAThave been replaced by valines. Although considering the possibility ofoxidation of cysteine in expressed rAAT in this study, variation ofmedium pH could induce the conformational or structural changes in theregion surrounding cysteine or reactive center loop (RCL), whichdominates the activity of binding target proteinase, and further alterthe functionality of human AAT for binding elastase. Additionally,although the cysteine residue of AAT could form a disulfide bridge withfree cysteine (Kolarich et al., 2006), the results show that both totaland functional human AAT are degraded in the cultured medium over time.Furthermore, the serine and cysteine protease inhibitor (AEBSF) help toretard the human AAT degradation in cultured medium (FIGS. 15, 16A and16B). Therefore, the proteolytic degradation of functional rAAT wasobserved and confirmed in this study. Consequently, the pH strategyproposed herein could both decrease protease activity and additionallystabilize the correct conformation of AAT for executing itsfunctionality.

The novel CMViva expression system, as described in the previousexamples, has been demonstrated that it could result in higherfunctional recombinant AAT production yield by transient expression inN. benthamiana leaves or transgenic N. benthamiana cell culture in abioreactor (see example 11) when compared with the expression levelsobserved using either the CaMV 35S constitutive promoter system or achemically estradiol-inducible, estrogen receptor-based promoter (XVE)system. This example proposes a pH condition-shifting culture strategyfor increasing the functional rAAT production yield (100 ng/mL) and theratio of functional rAAT to total rAAT (48%) in CMViva system. Thecomparisons of recombinant AAT production using CMViva system fromvarious systems are shown in Table 4. It apparently shows thatfunctional recombinant AAT production could be further improved by usinga pH shifting culture strategy in transgenic plant cell cultures,suggesting that the rAAT productivity by transgenic plant cell culturescould potentially compete with the levels achieved by transientagroinfiltration in plant leaves and furthermore the rAAT production isexposed to severe proteolytic degradation and rAAT protein is unstablein tobacco cell cultures.

Example 15 High-Level Transient Production of Heterologous Proteins inPlants by Optimizing Induction of a Chemically Inducible Viral AmpliconExpression System

The following example demonstrates two different methods of chemicaltreatment application, topical application and pressureinjection/infiltration, on heterologous protein production aredescribed. Details regarding construction of the CMViva expressionvector are described in previous examples.

Non-transgenic/wildtype N. benthamiana seedlings were grown from seed insoil-filled 10-cm pots. Seedlings were transplanted to individual 10-cmpots until plants were 10 to 15 cm in height, at which time plants wereready for infiltration. All plants were grown in a greenhouse with a16-hour photoperiod and a temperature range of 18° C. (nighttime low) to30° C. (daytime high). To prepare for evaluation of protein expressionin detached leaves, plant leaves were cut at the petiole and stored dryin unsealed plastic bags for one hour before infiltration to simulate asituation where plants would be harvested from a field and transportedto a facility for infiltration.

A. tumefaciens cells containing the appropriate plasmids were grown for24 hours in 2 mL of Luria-Bertani (LB) broth containing appropriateselection antibiotics. Approximately 0.5 mL was then transferred to 25mL of LB broth supplemented with 10 μL of 100 mM acetosyringone(3′,5′-dimethoxy-4′-hydroxyacetophenone) (Aldrich Chemicals, Milwaukee,Wis.) and 0.5 ml of 1 M 2-(4-morpholino) ethanesulfonic acid (MES)buffer (pH 5.6), and grown overnight at 28° C. with shaking until thecell density (optical density, OD600) reached 1.0 absorbance units.Cells were harvested by centrifugation at 2600 g, resuspended in 10 mLof sterile de-ionized water, and the cell density was adjusted to 1.0absorbance units. Five milliliters of the A. tumefaciens cell suspensionfor CMViva was mixed with 5 mL of A. tumefaciens cells containing thep35S:p19 plasmid, and then supplemented with magnesium chloride to reacha final concentration of 10 mM and acetosyringone to 150 □M, andincubated at room temperature for five hours. Bacterial suspensions werethen pressure infiltrated onto the abaxial side of either intact ordetached N. benthamiana leaves using a 3-mL sterile syringe without aneedle at three to four points to sufficiently cover the entire leaf(approximately 1 mL of bacterial suspension per 0.5 g fresh weight oftissue). No A. tumefaciens solution was applied to plants selected ashealthy control plants.

Intact plants that had been infiltrated were grown in the greenhouse forthe duration of the experiment. Leaves that had been detached werestored in a humidity chamber following infiltration to maintain thehealth of the plant material. The humidity chamber was prepared bysoaking 400 g of Perlite soil additive (E.B. Stone) in 1.5 L ofde-ionized water. Excess water was decanted and the saturated Perlitewas packed to form a 3-cm thick bed on the bottom of an 11.4 Lrectangular Tupperware container. Plant leaves were then placed 7 cmabove the Perlite bed on top of racks, and the chamber was then sealedand placed in the dark at 21° C. The humidity chamber was prepared fivehours before infiltration to ensure a humid environment uponintroduction of the plant leaves.

Twelve hours after infiltration, an induction solution composed of 50 μM17-β-estradiol (Sigma Inc., St. Louis, Mo.) in 0.05% Tween 20 wasapplied to all infiltrated N. benthamiana leaves using either cottontipped applicators to both sides of the leaves or a 3-mL sterile syringewithout a needle pressure injected onto the abaxial side of the leaves.To evaluate the effect of multiple inducer applications, the inductionsolution was applied to half of the total number of infiltrated leavesin the same manner (directly after sampling) at 2-day (48±1 h)increments after the initial induction. No inducer solution was appliedat any time to the healthy control plants.

Plant leaves were sampled on the infiltrated/induced areas 2 days (48±1h) post-induction (60±1 h post-infiltration) by collecting two 7-mmdiameter discs at various locations on each leaf and combining the discsto approximate an average leaf sample. Three leaves from differentplants were sampled for analysis of intact plant leaves and four leaveswere used for analysis of detached plant leaves. Plant leaves were alsosampled at 2-day (48±1 h) increments after the initial sampling (andafter additional inductions where applicable) to allow evaluation ofrecombinant protein production kinetics. Extraction buffer composed of20 mM Tris-HCl (pH 8.1), 150 mM NaCl and 0.01% (v/v) Tween 80 was addedimmediately after sampling at a ratio of 10 μL/mg fresh weight oftissue, and cells were lysed on ice using a plastic pestle. Lysate wascleared by centrifugation at 20,000 g for 20 min at 4° C. Thesupernatant was collected and stored at −80° C. until it was assayed.

The methods of protein quantification including Bradford assays fortotal soluble protein (TSP) analysis, total and functional AAT ELISAsfor recombinant AAT analysis were performed as described in the previousexamples.

The previous examples demonstrated that recombinant AAT can betransiently produced in non-transgenic N. benthamiana plants at highlevels using the CMViva expression system and that addition of the plantvirus-encoded gene silencing suppressor Tomato bushy stunt virus (TBSV)P19 gene is effective in enhancing productivity in the infiltratedleaves. In order to investigate if the transient production ofrecombinant AAT could be further increased, alternate methods were firstinvestigated for application of the chemical inducer. Non-transgenic N.benthamiana plant leaves were therefore co-infiltrated with A.tumefaciens cells containing CMViva and A. tumefaciens cells containingp35S:P19. Twelve hours after infiltration, leaves were induced either bytopical application of the induction solution via cotton tippedapplicators or by pressure injection via a needleless syringe. In orderto evaluate if additional inducer treatments improve productivity, theeffect of multiple inducer applications versus the traditional singleapplication was investigated. The kinetics of transient expression wereevaluated by sampling plants at two-day intervals for the duration ofthe experiment.

The total soluble protein (TSP) levels for plants exposed to each of thefour induction treatments as well as for the healthy uninfiltratedcontrol plants were measured on a fresh weight (FW) basis and are shownin FIG. 23. The data show that for each of the plants with inductiontreatments, the TSP levels are significantly higher than the maximum of8.4±0.20 mg TSP/g FW of tissue for the control plants. In each of theinduced plants, the maximum TSP levels are increased by as much as 72%compared to the control plants, resulting in a maximum of 14.4±0.73 mgTSP/g FW of tissue. For the control plants as well as each of the plantswith induction treatments, the TSP levels were at a maximum two daysafter induction, and steadily decreased to 56-60% of the maximum ateight days after induction. Based on the standard deviation of theaverages, there was no difference between TSP levels in plants that wereinduced via topical application and those that were injected.

ELISA analysis was used to quantify the amount of total rAAT transientlyproduced in each of the induced plants. The effects of inducerapplication methods on transient production of total rAAT in intactplant leaves were dramatic, as shown in FIG. 24. As expected, thehealthy control plant samples did not result in measurable rAAT levelsusing the highly specific ELSIA analysis. However, each of the inducedplants resulted in significant total rAAT production, with the maximumamount found at either six or eight days after induction. With regard tothe single topical application, the total rAAT level reached a value of1.4±0.17% of TSP at four days after induction, and increased to amaximum of 1.9±0.15% of TSP at six days after induction. The total rAATlevel was increased to 3.7±0.31% of TSP when using the pressureinjection method. When looking at the effect of multiple inductiontreatments, total rAAT levels are increased 120% and 57% compared tosingle treatment for the topical application and the pressure injectionmethods, respectively. A maximum total rAAT level of 5.8±0.46% of TSPwas obtained 6 days after induction for multiple induction treatmentsusing the pressure injection method.

When producing rAAT in plant hosts, it has been observed that not all ofthe rAAT produced is biologically active (see previous examples,Terashima et al. 1999; Huang 2001; Trexler 2002). The amount offunctional rAAT in each of the induced intact plants was quantified byELISA analysis. As shown in FIG. 25, the trends of the effects ofinducer treatment methods on transient production of functional rAAT inintact plant leaves mimicked the trends for total rAAT, except thatproduction of functional rAAT reached a maximum at four days afterinduction. The typical induction treatment of using a single topicalapplication of the chemical inducer resulted in a maximum functionalrAAT level 0.80±0.04% of TSP. Using the pressure injection method toapply a single application, a maximum level 1.8±0.09% of TSP wasproduced. The method of using multiple induction treatments increasedfunctional rAAT levels from 0.80±0.04% to 1.6±0.08% of TSP and from1.8±0.09% to 2.4±0.09% of TSP for the topical application and thepressure injection methods, respectively. It is also evident thatmultiple induction treatments help to maintain higher levels offunctional AAT over longer time periods as greater than 75% of themaximum levels are still present at six days after induction while lessthan 40% of the maximum remains when only a single induction treatmentis applied. Functional rAAT was undetectable in the control plants usingthe ELISA method.

The production kinetics of the functional rAAT and total rAAT levelsdiffer in the intact plant leaves, hence the timing of the maximumfunctional rAAT and maximum total rAAT levels do not occur on the sameday. Therefore, functional rAAT production using each of the inductiontreatments methods was evaluated as a percentage of the total rAATproduced. The percentage of total rAAT that is biologically active ishighest early in the production process, as evidenced by FIG. 26. Forall induction treatments the percentage of functional rAAT was highesttwo days after induction. Topical application of the inducer resulted in61-65% functional product while pressure injection increased levels to83-90%. While multiple inductions did not increase the maximumpercentage of functional rAAT, the percentage of functional rAATremained higher at six and eight days after infiltration. By the eighthday after induction however, less than 15% of the total rAAT wasfunctional for any of the treatments.

In order to evaluate transient expression of a recombinant humantherapeutic protein using a platform that is more amenable to scale-upthan intact plant leaves and that would circumvent the need toinfiltrate plants in the field or the requirement of a specializedgreenhouse facility, rAAT production in detached N. benthamiana leaveswas investigated. Plant leaves were detached from whole plants andstored dry for about one hour before infiltration. Detached leaves fromnon-transgenic N. benthamiana plants were then co-infiltrated with A.tumefaciens cells containing CMViva and cells containing p35S:P19.Twelve hours after infiltration, detached leaves were induced using thesame treatment methods as the intact plants to evaluate the effects oftopical application versus pressure injection, a single applicationversus multiple applications, and the production kinetics were againevaluated in the same manner as before. Infiltrated and induced plantleaves were stored in a humidity chamber throughout the course of theexperiment. This production scheme was used to simulate a productionroute where non-transgenic plants would be harvested from a field andtransported to a facility for infiltration and subsequent induction.

Total Soluble Protein Production (TSP) levels for detached leaves weremeasured and are shown in FIG. 23. Again, TSP levels are significantlyhigher in the induced plants than in the control plants. In all cases,TSP levels were at a maximum early in the production process, anddecreased throughout the experiment. The maximum amount of TSP for thecontrol leaves was 3.4±0.18 mg TSP/g FW of tissue. Detached leavessubject to topical application of the inducer resulted in the highestTSP levels, 6.4±0.08 and 6.5±0.28 mg TSP/g FW of tissue for single andmultiple applications, respectively. For both single and multipleapplication treatments, the injected leaves resulted in lower maximumlevels of TSP than for the topical application. Comparing TSP levels indetached leaves to intact leaves shows there is generally 50% less TSPon a per FW basis in the detached leaves at all times.

FIG. 24 illustrates the effects of inducer treatment methods ontransient production of total rAAT in detached plant leaves. For allinduced plants, maximum levels of total rAAT were obtained anywherebetween four and six days post induction. Comparing the singleapplication to the multiple application leaves when using the topicalmethod shows an increase from a maximum value of at 2.5±0.08% to3.5±0.15% of TSP. Single application treatment using pressure injectionresults in 2.4±0.18% of TSP, which is equal to within one standarddeviation of the total rAAT level obtained from the single topicalapplication. However, leaves exposed to multiple pressure injections donot show increased levels of functional rAAT. It is important to pointout here that in detached leaves induced using the syringe method, ringsof necrotic regions caused by applying high pressure on the luer tip ofthe syringe to the surface of the leaf were observed around the site ofinjection. The areas within and around these injection sites started tothin and turn brown in color by the third to fourth day after induction.This observation was even more evident when multiple inductions wereused, as there were more regions of necrosis caused by the additionalinjections. On a percent TSP basis, total rAAT production in detachedleaves compares well to that in intact leaves for the topicalapplication method, even surpassing the maximum level by 0.6% of TSPwhen a single application was used.

FIG. 25 shows the levels of biologically functional rAAT transientlyproduced in detached leaves. Here, the production kinetics shows amaximum amount of functional rAAT at four days post induction. Thetrends for topical applications are similar to those described for totalrAAT as multiple applications results in much greater rAAT levels thanthose obtained from a single application, 1.3±0.05% and 0.71±0.04% ofTSP at four days post induction, respectively. Single applicationtreatment using pressure injection results in 1.3±0.06% of TSP, 80%higher than using a single topical application. Once again the syringeinjection method reduced the total rAAT levels when multipleapplications were applied, presumably due to tissue damage. For thetopical application method, functional rAAT production in detachedleaves was only slightly less than for intact leaves (0.09 and 0.3% ofTSP less than in intact leaves at four days post induction for singleand multiple applications, respectively).

The percentage of biologically functional rAAT to total rAAT fordetached leaves is displayed in FIG. 26. For each of the inductiontreatment methods, the percentage of functional rAAT was highest at twodays after induction. For the topical application method, the maximumpercentage of functional rAAT was 55-65%, while for the injection method85-87% was found to be biologically active. The number of inducerapplications did not appear to enhance stability of functional rAAT. Themaximum percentage of rAAT for each of the induction application methodsin detached leaves was within 10% of the values obtained for intactleaves.

While many methods are used for chemical induction solution to plantsleaves, most fall into one of the two distinct categories of eitherusing a surface application or using a pressure gradient driving forceto apply the solution. To evaluate the differences in protein productionbetween these two categories we elected to evaluate productionstrategies that would be representative of the maximum amount of inducersolution that could be applied to the plant leaves using any of thecurrent induction methods, many of which can be used for scale-up. Forexample, topical application via a cotton swab represents the maximumamount of inducer that could be applied to the surface of a plant leafusing any of the surface application methods (such as dipping). Pressureinjection using a syringe without a needle represents the maximum amountof induction solution that could be driven into the tissue using apressure driving force induction strategy (such as vacuum infiltration).Our results show that in intact plants using a single pressureinjection, we were able to dramatically increase heterologous proteinproduction as we obtained 95% and 125% more total and functional rAAT,respectively, compared to production using a single topical application.These results suggests that the topical application of the inductionsolution either does not reach all of the infected cells or the amountof induction solution reaching the cells in the center of the leaftissue is not adequate for optimal activation, as induction has beenshown to be concentration dependant (Zuo et al. 2000; Sun et al. 2003).This concern is critical whether using stably transformed plants ortransient expression and should be addressed when attempting to achieveoptimal induction using a chemical inducible promoter system in planttissues.

Previous examples show that the number of inducer applications cangreatly impact protein expression. Previous research has shown thattranscript levels and subsequent recombinant protein production reach amaximum and then decrease over time when using inducible expressionsystems and suggests that modifications to the current inductiontreatment process may be necessary for optimal protein production (Zuoet al. 2000; Moore et al. 2006; Dohi et al. 2006). To address thisissue, we first investigated if we could keep transcript levels high andincrease protein production by applying additional inducer. We electedto add induction solution every two days as previous research has shownwhen using the XVE expression system as used in this study, transcriptlevels reach a maximum and then decrease two to four days afterinduction (Zuo et al. 2000; Dohi et al.). When comparing productionusing multiple applications to the more conventional method of a singleapplication of induction solution, our results show that maximumfunctional rAAT expression levels were increased by at least 35% in bothtreatment methods in intact plants and for topical induction in detachedplant leaves, as shown in FIG. 23. We were able to maintain the amountof functional rAAT for a longer time period using multiple inductions as75% of the maximum level is still produced six days after induction. Ourresults suggest that in situations where only a single application isused, the inducer may become limiting due to uptake and binding ofestradiol to the XVE protein or degradation of estradiol, as has beenobserved with other chemical inducers (Moore et al. 2006). As previouslymentioned, the results using syringe injection in detached leavesdeviate from the expected trend since pressure injection compromisedplant leaf health. In addition, this effect was amplified when applyingmultiple injections.

As the production levels in plant-based systems continue to increase,the economics may favor a switch from traditional production methods toplant-based systems as is increasingly evident by the number ofcompanies advancing such proteins through clinical trials (Nykiforuk etal. 2006; Fox 2006). Therefore, scale-up of the process will become animportant issue. Using a strategy that mimics harvesting non-transgenicplant biomass from a field or greenhouse and bringing it to a facilityfor production, our data show that production in detached leaves resultsin approximately half of the overall TSP obtained with intact leaves.This lower amount is expected since detached leaves no longer have asource of nutrients to sustain production. However, as portrayed in FIG.25, production in detached leaves results in only slightly lessfunctional rAAT than the intact plant leaves, on a TSP basis. When usingmultiple surface induction treatments, the resulting production indetached leaves is 1.3±0.05%, very nearly approaching the value of1.6±0.08% of TSP obtained form intact plants. Although the functionalrAAT levels for detached leaves are similar to intact leaves on % TSPbasis, because the TSP levels in detached leaves are significantlylower, the functional rAAT levels are lower per gram FW tissue than forthe intact leaves. For detached leaves, pressure driven applicationmethods may need to be altered to approach production levels achievablein intact plants.

Chemically inducible promoters, especially utilizing viral ampliconexpression systems, are showing great promise for rapid high-levelproduction of heterologous proteins in plant hosts. In this study, wehave demonstrated that the method of the inducer application can be usedto dramatically increase the amount of heterologous protein produced inchemically inducible agroinfiltrated systems. Combining both thestrategies of multiple applications and pressure infiltration in intactleaves, production of heterologous protein was increased by nearly3-fold to levels to nearly 2.5% of the TSP for biologically functionalprotein and nearly 6% of the TSP for overall heterologous protein. Thisis a significant improvement over the traditional single topicalapplication. The highest reported level of recombinant proteinproduction using a chemically inducible viral amplicon expression systemhas been the production of the reporter protein, GFP, which has beenexpressed at up to 10% of the TSP (estimated by band intensity) fourdays after induction in a stably transformed tobacco suspension cultureusing the XVE promoter driving a tomato mosaic virus (ToMV) viralampicon expression system (Dohi et al. 2006). Our production of aheterologous human protein at almost 6% of TSP compares well toproduction of this report protein, especially considering that AAT isdegraded in plants (Terashima et al. 1999) while GFP is known to be verystable. Production of heterologous protein using our expression systemcompares favorably to production using the bean yellow dwarf virus(BeYDV) alcohol inducible expression system, in which production ofNorwalk virus capsid protein (NVCP) has been demonstrated up to 1.2% ofTSP in a stably transformed tobacco suspension culture (Zhang et al.2006). To date, production using the CMViva system is the only report oftransient heterologous protein production using a chemically inducibleviral amplicon expression system.

Studies have shown that the production of rAAT in plant hosts results inboth active and inactive form of rAAT being produced (Terashima et al.1999; Huang et al. 2001; Trexler et al. 2002). The same observation wasobserved in this example, as is evident in the larger amounts of totalrAAT than functional rAAT. For intact plants, the maximum level ofproduction is 5.8±0.46% of TSP for total rAAT and 2.4±0.09% of TSP forfunctional rAAT. When looking at the relative percentage of functionalto total rAAT, it is evident that functional protein is being diminishedsince functional rAAT is greater than 50% for topical and 85% forpressure induction application after only two days of production, butthen quickly decreases throughout the production process. The same istrue for production in detached plant leaves. This suggests that eitherprotein denaturation/unfolding or protein degradation/cleavage isoccurring, as has been observed in the past for plant-produced rAAT(Terashima et al. 1999; Huang et al. 2001; Trexler et al. 2002).However, this also suggests that optimizing the amount of rAAT producedduring the early stages of production may lead to a higher overall yieldof functional rAAT. Alternately, if degradation can be reduced such thatthe maximum percent of functional rAAT can coincide with the maximumlevel of total rAAT production, then it would be possible to achieve5.8±0.46% of TSP as biologically functional rAAT using multiple syringeinduction treatments in intact N. benthamiana leaves or 3.5±0.15% of TSPwhen using detached plant tissue. It was also observed that the totalrAAT levels do not drop off as rapidly as functional rAAT levels, so ifdegradation can be reduced not only could very high yields be achievedbut the maximum level of rAAT production would be less sensitive to thetime at which the protein is recovered.

High-level transient production of heterologous proteins can be achievedin both intact and detached plant leaves by utilizing a chemicallyinducible viral amplicon expression system. Altering the method in whichthe induction solution is applied in order to increase contact to theplant cells can further elevate production. By applying inductionsolution via topical application to mimic total surface coverage andapplying the inducer every two days to infiltrate detached plant leaves,maximum levels of biologically functional rAAT were increased 1.8-fold.When evaluating production in detached leaves, it is particularlyimportant to ensure the method of induction does not compromise thehealth of the leaf and, therefore, multiple pressure driven injectionsare not recommended for detached leaves although it is noted that analternate driving force method that does not damage the plant tissue mayresult in higher production. Production can be elevated to an evenlarger degree using alternate induction strategies in intact leaves. Byapplying multiple applications of induction solution via pressureinjection to infiltrated intact leaves which provides the most uniformand constant inducer concentration within the tissue, maximum levels ofbiologically functional rAAT were elevated up to 3-fold compared to theconventional method of a single topical application, and expressionlevels remained high even six days post induction. Therefore, forproduction in intact plant leaves, multiple application of inductionsolution using a driving force method is recommended to increase inducercontact and availability. This example lays the basis for developinginduction strategies to scale-up processes which use chemicallyinducible viral amplicon systems in order to achieve high-leveltransient production of heterologous proteins in N. benthamiana plantleaves.

Various modifications and variations of the present invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of theclaims.

REFERENCES

The following references cited herein are hereby incorporated byreference in their entirety.

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TABLE 1 Primer Sequence Description O14G10Pf5′-ACATGCAATGCATCTGATAGTTTAAACTGAAGGC-3′ Forward primer for SEQ ID NO: 1G-10-90 promoter O15NPeaTr 5′-ACAGGATGCGGCCGCCAGTTTCCAAGCTTGTTTG G-3′Reverse primer for SEQ ID NO: 2 G-10-90 promoter O16NOpf5′-CATACCAATGCATTTACTTGCACAGCTTGG-3′ Forward primer SEQ ID NO: 3for OlexA operator O17XSOpr 5′-CATATCTCGAGGCTAGAGTCGAC-3′ Reverse primerSEQ ID NO: 4 for OlexA operator OQCD3-15′-ACTTCTCGAGTGGCGCGCCGGTCAACATGGTGGAGC-3′ Forward primer SEQ ID NO: 5for 35S promoter OQCD3-2 5′-ACACGAATTCAGGCACACTGAGACGCA-3′Reverse primer SEQ ID NO: 6 for CMV RNA3 OQCD3-35′-AATGAAGCTTAATTCCTATCTCACGGATG-3′ Forward primer SEQ ID NO: 7for CMV RNA3 OQCD3-4 5′-ATCTGGATCCTGGTCTCCTTATGG-3′ Reverse primerSEQ ID NO: 8 for CMV RNA-3 OQCD3-5 5′-ATACGGATCCATTCGGTACGCTGAAATC-3′Forward primer SEQ ID NO: 9 for 35S-T in pQCD3 OQCD3-65′-ATGTAGATCTGGCGCGCCGGATTTTAGTACTGGAT-3′ Reverse primer SEQ ID NO: 10for 35S-T in pQCD3 OSDAATPst 5′-TCAACTGCAGAACAATGAAGAACACCTCCTCCCT-3′Forward primer SEQ ID NO: 11 for SD-AAT OSDAATCPst5′-ACATCTGCAGTCACTTCTGCGTGGGGT-3′ Reverse primer SEQ ID NO: 12for SD-AAT

TABLE 2 Comparison of N. benthamiana growth and rAAT production inbioreactors using different expression systems Maximum Maximum MaximumMaximum Extracellular OUR_(max) Extracellular ExtracellularExtracellular X_(o) X_(max) μ_(max) Protease Activity (mmol O2/ TotalrAAT Functional Functional/Total System (g-DCW/L) (g-DCW/L) (day⁻¹)(U/L) L-h) (μg/L) rAAT (μg/L) rAAT (%) Wild type 1.4 13.4 (d 9)* 0.26 981 (d 10) 1.62 (d 8)  — — — 35S 0.9 12.2 (d 22) 0.14 1610 (d 22) 0.90(d 16)  3500 (d 22)  5.9 (d 22) 0.17 (d 22) XVE 1.2 12.3 (d 10) 0.242550 (d 20) 1.2 (d 11) 690 (d 22) 4.1 (d 19) 0.93 (d 19) CMViva 1.5 12.7(d 10) 0.26 3420 (d 18) 1.4 (d 10) 140 (d 16) 27.3 (d 18)  21.9 (d 18)(day)*day after inoculation. For XVE and CMViva system, rAAT was inducedby adding inducer at day 11 after inoculation.

TABLE 3 Comparison of rAAT production in N. benthamiana with the CMVivasystem following induction Func- tional Total Functional FunctionalTotal rAAT/ rAAT/ rAAT/ rAAT/ rAAT/ TSP^(a) TSP Total DCW^(b) DCW System(%) (%) rAAT (%) (%) (%) Transgenic 0.019 0.086 22 2.4 × 10⁻⁴ 1.24 ×10⁻³ plant cell cultures in bioreactor, without p19* Transient 0.16 0.5728 2.5 × 10⁻² 9.1 × 10⁻² expression in whole intact plant leaves,without p19** Transient 1.2 1.7 71 1.6 × 10⁻¹ 2.38 × 10⁻¹  expression inwhole intact plant leaves, with p19** ^(a)TSP = Total Soluble Protein^(b)DCW = Dry cell weight *this example **previous examples

TABLE 4 Functional Functional Total rAAT/Total rAAT/TSP^(a) rAAT/TSPrAAT System (%) (%) (%) CMViva, transient 0.16 0.57 28.1 expression inwhole plant leaves, w/ induction, w/o p19 CMViva, transient 1.2 1.7 70.5expression in whole plant leaves, w/ induction, w/ p19 CMViva,transgenic 0.019 0.086 22.1 plant cell cultures in bioreactor, w/induction, w/o p19, (extracellular rAAT yield) CMViva, transgenic 0.0660.137 48.2 plant cell cultures in bioreactor, pH control, w/ induction,w/o p19, (extracellular rAAT yield)

We claim:
 1. A plant expression system for the expression of aheterologous gene in a plant or plant cell culture, the plant expressionsystem comprising a cDNA encoding a CMV1a replicase and a cDNA encodinga CMV2a replicase, wherein the cDNA encoding the CMV1a replicasecomprises a CMV RNA 1 cDNA with a 5′ nucleotide sequence deletionconsisting of deletion of 57 nucleotides from the 5′ end of the CMV RNA1 cDNA, wherein said nucleotide sequence deletion prevents replicationof CMV1a transcripts when a functional replicase is present, and whereinthe cDNA encoding the CMV1a replicase is operably linked to a LEXoperator and expression of the CMV1a replicase is estradiol-inducible.2. The expression system of claim 1, wherein the heterologous gene codesfor a human protein.
 3. A plant or plant cell comprising the plantexpression system of claim
 1. 4. The expression system of claim 1,wherein said cDNA encoding said CMV2a replicase is operably linked to aCaMV 35S promoter.
 5. The expression system of claim 1, wherein theheterologous gene is operably linked to a CaMV 35S promoter.
 6. Theexpression system of claim 1 further comprising a cDNA encoding a genesilencing suppressor.